Download Treatment of patients with non-Hodgkin lymphoma and lactic acidosis

Melanie van Berkum
July 2011
Name of thesis:
Non-Hodgkin lymphoma, its side-effects and the involvement of the
IGF-system
Made by:
Supervisor:
Education:
Date:
Melanie van Berkum
Dr. Jaap van Doorn
Biomedical Sciences
April-July 2011
Explanation front page
This thesis is about non-Hodgkin lymphoma, a diverse group of different types of blood
cancer, which are primarily caused by abnormal B-lymphocytes, which can become
cancerous. In this picture a B-lymphocyte is shown, with his B-lymphocyte specific proteins
on his surface.
Reference
http://www.biooncology.com/therapeutic-targets/cd20/index.html
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
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Index
Rationale……………………………………………………………. p. 4
Chapter 1: Acidosis………………………………………………… p. 5-15
1.1: General metabolic acidosis …………………………………………
1.2: Ketoacidosis…………………………………………………………
1.3: Lactic acidosis………………………………………………………
1.4: Effect of lactic acidosis on the growth hormone (GH)/insulin like
growth factor-I axis………………………………………………
1.5: Relationship between lactic acidosis and hypoglycaemia……...…...
1.6: Diagnosis of cancer by the Warburg effect………………………….
1.7: Treatment of patient with non-Hodgkin lymphomas and lactic
acidosis……………………………………………………………
p. 6
p. 6-13
p. 13
p. 13
p. 13-14
p. 14
p. 14-15
Chapter 2: Non-Hodgkin Lymphoma (NHL)…………………….. p. 16-20
2.1: Diagnosis……………………………………………………………. p. 16-18
2.2: Pathology……………………………………………………………. p. 18
2.3: Different types and their treatment………………………………….. p. 18-20
Chapter 3: The IGF-system……………………………………….. p. 21-32
3.1: The growth hormone-releasing hormone-growth hormone IGF-axis.
3.2: IGF-I and IGF-II receptors…………………………………………..
3.3: Effects of IGF-I and IGF-II………………………………………….
3.4: IGF-binding proteins (IGFBPs) and the acid labile subunit (ALS).…
3.5: The IGF-system under pathological conditions……………………..
p. 21
p. 22-24
p. 24-25
p. 25-28
p. 28-32
Chapter 4: The IGF-system, lactic acidosis and leukaemia (NHL):
Case reports……………………………………………….….. p. 33
Chapter 5: Various components of the IGF-system in a patient
with non-Hodgkin lymphoma and lactic acidosis…...…….. p. 34-42
References………………………………………………………….. p. 42-45
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Rationale
A patient with Non-Hodgkin lymphoma and acidosis was admitted to the Reinier de Graaf
Gasthuis in Delft. Serum samples of this patient were send to the Laboratory of
Endocrinology of the University Medical Centre Utrecht for further analysis of components of
the insulin growth factor (IGF) system. At present only a few case reports appeared that
described the possible involvement of the IGF system in patients suffering from NHL and
either acidosis and/or hypoglycaemia. In fact, this was the reason for the subject of the present
thesis.
(NHL) is a malignant disease which represents 3-5% of all cancers registered in Europe. It is
caused by a dysfunction of the B-lymphocytes, what results in lymphadenopathy and other
symptoms1. A rare complication of NHL is the occurrence of lactic acidosis and/or
hypoglycaemia.
In this thesis the literature on the involvement of the IGF system in NHL and acidosis and
hypoglycaemia is reviewed. During the brief internship in the Laboratory of Endocrinology
various components of the IGF system in patient’s sera, as obtained both before and during
treatment, were investigated. The possible implications of the results are discussed.
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Chapter 1: Acidosis
1.1: General metabolic acidosis
Metabolic acidosis can be defined as a process that leads to a disturbance in the homeostasis
of the acid-base balance, i.e. acidification, in body fluids. In general, acidosis is said to occur
when arterial pH falls below 7.35. It is induced by a decrease in concentration of bicarbonate
in body fluids. This can be caused by the ingestion of an excess of acid, an overproduction of
organic acids, or a decreased elimination of acid H+ by the kidney. Another possible cause for
metabolic acidosis is the excessive loss of base, for example in the case of severe diarrhoea2.
Metabolic acidosis will result in immediate lowering of the pH and an increase of the
concentration CO2 in the circulation. As a consequence, patients will start to hyperventilate in
order to increase the pO2 in the blood and to increase the pH back to its normal level. When
metabolic acidosis continues, i.e. more than 3 hours, the kidney is stimulated to excrete more
ammonium which leads to increased efflux of protons in distal parts of the nephron. Chronic
metabolic acidosis is the situation with a lower plasma bicarbonate concentration2.
Effects of metabolic acidosis on protein metabolism, nitrogen balance, and levels of
calcium and phosphate
Acidosis has a negative effect on the nitrogen balance, i.e. relatively more nitrogen and
proteins are found in the urine. Probably this is caused by an increased activity of
glucocorticoids. Glucocorticoids stimulate mRNA expression of ubiquitin-proteasome genes,
which mediate the proteolysis of especially muscle proteins.
Also other endocrine mediators, e.g. the thyroid hormones, contribute to alterations in protein
metabolism and thus nitrogen balance. It increases protein breakdown and stimulates
branched amino acid to oxidize. Also protein synthesis is inhibited2.
Metabolic acidosis also induces hypercalciuria, by several mechanisms. Firstly, acidosis
increases the release of calcium from bone. Secondly, there is a decrease in the reabsorption
of calcium.
In addition, metabolic acidosis increases clearance of phosphate; phosphate clearance is
increased in the kidneys and fractional excretion is decreased2.
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1.2: Ketoacidosis and alcoholic acidosis
Ketoacidosis is a metabolic state associated with high concentrations of ketone bodies in the
circulation, due to the accelerated breakdown of fatty acids by the liver and the deamination
of amino acids. The two common ketones produced in humans are acetoacetic acid and βhydroxybutyrate. An excess of these ketone bodies may significantly acidify the blood. This
phenomenon is most common in the case of untreated diabetes when the beta cells of the
pancreas produce insufficient insulin to increase glucose uptake and to inhibit the hormone
sensitive lipase. Besides diabetic ketoacidosis, another form, alcoholic acidosis, may occur. In
that case, exessive alchohol consumption causes dehydration and blocks the first step of
gluconeogenesis. The body is unable to synthesize enough glucose to meet its needs, and, as
in diabetic acidosis, leading to a lack of energy resulting in increased fatty acid metabolism,
and subsequent body formation3.
1.3: Lactic acidosis
A special form of acidosis is lactic acidosis, which is caused by an overproduction of lactate.
Lactate is the end product of anaerobic glycolysis, and 90% of all lactate will pass through the
liver where it will be converted into pyruvate and later on into glucose. The remaining 10% is
cleared by the kidneys.
When these two processes are deregulated, lactic acidosis will develop. Lactic acidosis can
be diagnosed by low pH (<7,35) and an accumulation of lactate in the blood (>5 mmol/L)
There are two types of lactic acidosis. Type A is caused by tissue hypoxia or hypoperfusion,
when no oxygen is delivered to the tissues. The pH decreases and the cells metabolize glucose
anaerobically, which leads to an overproduction of lactate. Type A is more common then
Type B (see below), which is most related with haematological malignancies like leukaemia
and lymphomas3. Several patients with lactic acidosis also have been diagnosed with
hypoglycaemia, which is due to a high rate of glycolysis4. Cancer cells can grow so fast that
they outgrow their blood supply, and thus create a great production of lactate5.
Patients with lymphomas and lactic acidosis are often very ill, and for that the reason it is
sometimes hard to determine whether lactic acidosis is only the result from the malignancy or
also has another cause3.
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Warburg effect
In this part we focus on the Type B, caused by e.g. leukemia and lymphomas. This type of
acidosis is thoroughly investigated by Nobel Prize winner Otto Warburg. In the 1920s, he had
shown that most cancer cells have an aberrant energy metabolism.
Normal cells obtain their energy by transferring glucose into pyruvate by glycolysis. Pyruvate
enters the mitochondria, where it is oxidized to water and carbon dioxide. Via oxidative
phosphorylation adenosine triphosphate (ATP) is formed, the most important form of energy.
This process needs oxygen.
Aerobic glycolysis requires enough NAD+ (nicotinamide-adenine dinucleotides), what means
that NADH has to be reoxidized, conform the following reaction:
NADH + H+ + coenzyme Q  NAD+ +QH2
This reaction occurs in the mitochondrial membrane by oxidative phosphorylation. For this
reaction coenzyme Q is needed. After the protons are transferred to this coenzyme, it is named
ubiquinol. Because this reaction is a step of the oxidative phosphorylation, ATP will be
produced. Since cancer cells are adapted to hypoxic conditions, this reaction occurs at a much
lower rate than in normal cells, even in the presence of sufficient amounts of oxygen.
Moreover, cancer cells usually contain less mitochondria, thus also for this reason less ATP
can be produced by the oxidation of pyruvate.
Because in glycolysis only two molecules ATP are produced per glucose molecule, the cancer
cells have to accelerate the glycolysis pathway in order to full fill their energy requirements
Hence, glycolytic formation of pyruvate will occur at a faster rate (up to 200 times than in
normal cells).This phenomenon is called the Warburg effect. This effect results in a higher
production of lactate, whereby NADH will be reoxidized to NAD+ by lactate dehydrogenase .
Pyruvate + NADH + H+  Lactate + NAD+
Lactate cannot be taken up and metabolized by muscle, and for that reason lactate will diffuse
into the blood, and transported to the liver. Here, it is converted to glucose via
gluconeogenesis. Pyruvate kinase, a mitochondrial enzyme, plays an important role by
forming oxaloacetate from lactate67.
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Fig. 1
6
Metabolism of lactate: M = mitochondria; C = cytosol; AcCoa = Acetyl coenzyme A; TCA = Krebs
cycle; OxAc = oxaloacetate
The Warburg effect may provide other advantages for neoplastic cells.
Proliferating cancer cells need a lot of carbon precursors for biosynthesis. These are formed
during the glycolysis and in the pentose phosphate pathway. The Warburg effect includes a
high rate of glycolysis, and thus forms enough precursors needed for biosynthesis. Another
consequence of the Warburg effect involves the protection and invasion of the tumour.
As emphasized previously, cancer cells produce high amounts of lactic acid that is transported
out of the cells and causing an acid environment. Tumour cells are resistant against a low pH,
but normal human cells are not. The latter are destroyed by the low pH. In this way, acidosis
protects the tumour cells also against cells of the immune system7.
Additional causes of lactic acidosis
Deficiencies of vitamins
Thiamine deficiency
Thiamine (vitamin B1) is a cofactor of pyruvate dehydrogenase, which converts pyruvate into
acetyl coenzyme A (fig. 2). When thiamine is deficient, pyruvate is predominantly converted
to lactate3.
It is still unclear whether thiamine deficiency is the cause of lactic acidosis or not. In some
patients with lymphomas and lactic acidosis, the patient was supplemented with thiamine, but
this treatment did not diminish lactic acidosis significantly3. In another study, a patient with
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lactic acidosis, received a vitamin cocktail that contained vitamin B12. After this cocktail, the
patient improved dramatically8.
Fig. 2
8
The role of TPP (thiamine pyrophosphate) in the metabolism of glucose. When TPP is not present,
pyruvate can not form Acetyl-CoA and thus pyruvate stays in the cytosol and will form lactate.
Biotin
Biotin (vitamine B7) is a cofactor of different carboxylases. Therefore, it plays an important
role in the metabolism of the cell. It is recycled by biotinidase, and thus intake by nutrition
does not have to be high. Biotin deficiency can be caused by inherited disorders. Patients with
such a disorder show high levels of 3-hydroxypropionate and 3-methyl-crotonil-glycine, two
products
of
the
biotin-dependent
carboxylases:
propionyl-CoA
carboxylase
and
methylcrotonyl-CoA carboxylase. These enzymes catalyze reactions in gluconeogenesis, and
thus play an important role in metabolic homeostasis89.
Liver and/or kidney dysfunction
Because the fact that lactate should be cleared by the kidneys and by the liver (to be
metabolized into pyruvate and in part being converted into glucose), it has been hypothesised
that liver and/or kidney dysfunction can cause an accumulation of lactate in patients with nonHodgkin lymphoma. This is because cancer cells can infiltrate into these organs leading to
liver and/or kidney dysfunction10.
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This hypothesis, however, has been challenged recently. Patients with lymphoma and lactic
acidosis indeed showed larger kidneys that were infiltrated with leukaemia cells. However,
function of the kidneys was not impaired3.
When liver dysfunction occurs, the liver utilizes less lactate via the gluconeogenesis pathway,
which causes an accumulation of lactate. This hypothesis may also be invalid, since patients
with liver cirrhosis or hepatic failure, in the absence of malignancy, did not show signs of
lactic acidosis10.
Microembolism
Leukaemia and lymphomas may cause microembolism by the formation of microthrombi.
This leads to ischemia in the tissue and anaerobic glycolysis, which results in the production
of lactate. However, it is not likely that thrombosis is a major cause of lactic acidosis, because
never a relationship between thrombosis and lactic acidosis has been demonstrated3.
Overexpression of hexokinase
The primary goal of tumour cells is to proliferate, and therefore they need enough energy and
phosphometabolites. As emphasized previously, for this reason, tumour cells increase their
glycolytic rate. One example of achieving this is by the overexpression of glycolytic enzymes.
In the glycolysis three enzymes have the ability to be rate limiting10.
One of those enzymes is hexokinase II, the first rate limiting enzyme in the glycolytic
pathway, which is primarily present in adipose and muscle tissue. It transfers glucose into
glucose-6-phosphate. It has a high affinity for glucose, what means that the enzyme is all
ready active when glucose levels in blood are low. Voltage-dependent anion channel (VDAC)
binds to hexokinase II and initiates the activity of the enzyme and stimulates the glycolysis.
On the other hand, this interaction is critical to prevent the induction of apoptosis in tumour
cells11.
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Fig. 3 11The conversion of glucose to lactate. Glucose is transported into the cell via glucose transporter (Glut).
In the mitochondrion ADP is transposed to ATP by ATP synthase and adenine nucleotide transporter transports
ATP to the voltage-dependent anion channel (VDAC). This binds hexokinase II, that phosphorylates glucose
into glucose-6-phosphate by the use of ATP. This can be used for the pentose phosphate pathway, for synthesis
of nucleic acids. In malignant diseases however, glucose-6-phosphate is used to form lactate.
B-cell lymphoma (Bcl)-2
In normal cells, apoptosis is needed for tissue homeostasis. The Bcl-2 family of proteins
consists of pro-apoptotic proteins and anti-apoptotic proteins, both located in the outer
mitochondrial membrane of intact cells.
Pro-apoptotic proteins can cause swelling of the mitochondria and disruption of the outer
mitochondrial membrane by promoting loss in mitochondrial membrane potential (Δψm). For
this reason, the content of the mitochondria can leak into the cytoplasm. One example for this
is cytochrome c, an essential component of the oxidative phosphorylation in the mitochondria.
VDAC might be the reason for the increased permeability of the mitochondrion. When it
binds to hexokinase II, the mitochondrial permeability transition pore complex (MPTP) is
inhibited. When VDAC is absent, this complex is not inhibited and cytochrome c will leak
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into the cytoplasma. Oxidative phosphorylation becomes impaired. Thus, when pro-apoptotic
proteins are overexpressed, the metabolism of the cell is changed. Another result of the loss of
mitochondrial membrane potential is a reduction in mitochondrial ATP production. Normally,
ATP inhibits glycolysis by inhibiting phosphofructokinase, but because of the low
concentration of ATP, glycolysis is stimulated, especially anaerobic glycolysis.
In summary Bcl-2 proteins trigger apoptosis by a loss of mitochondrial membrane potential,
what results in an increased lactate production11,12.
On the other hand, the Bcl-2 proteins also prevent acidification by inhibiting proton flux out
of mitochondria, to prevent mitochondrial dysfunction. Acidification is also prevented by the
vacuolar H+-ATPase. This pump pumps protons out of the cytoplasm into endosomes or the
extracellular space12. The exact, net function of Bcl-2 remains unclear.
TNF-alpha
Tumour necrosis factor-alpha (TNF-alpha) is a cytokine which has an important role in the
immune system. For this reason, it also plays a paracrine role against tumour cells, especially
against the mitochondrial function.
It causes a reduction in the activity of pyruvate dehydrogenase, which results in a decreased
conversion of pyruvate into acetyl-coA. A high level of pyruvate arises, and in turn it will be
converted into lactate.
The second effect of TNF-alpha is inhibiting the cytochrome-dependent electron transport
system. As a consequence, that NADH cannot be reoxidized, and must be obtained from
pyruvate through the action of lactate dehydrogenase wherby lactate is formed.
Both effects results in a higher level of lactate and thus contribute to lactic acidosis10.
Therapies
Methotrexate
The development of lactic acidosis may also be the result of chemotherapy. Chemotherapy
contains methotrexate, what competes with the reduced folate carrier (RFC-1), a carrier in the
thiamine transport system. When this carrier is blocked, the transport of phosphorylated
thiamine derivatives into the cells is hampered. In this way, less thiamine is available, what
results in the same mechanism of disease as in patients suffering from thiamine deficiency108.
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Nucleoside reverse transcriptase inhibitors
Patients with human immunodeficiency virus (HIV) are treated with nucleoside reverse
transcriptase inhibitors. These inhibitors lead to aberrant glycolytic processes by
mitochondrial dysfunction that stimulates anaerobe glycolysis10.
Tumour lysis syndrome
As long as the overproduced lactate is not readily released to the extracellular space, the cell
will go into apoptosis, and no lactic acidosis will occur in the blood. Non-Hodgkin
lymphoma, however, goes often together with acute tumour lysis syndrome (ATLS), being
characterized by a release of intracellular contents into the bloodstream, which leads to
metabolic complications12.
1.4: Effect of lactic acidosis on the growth hormone (GH)/insulin-like
growth factor-I axis
It has been shown that subjects with acidosis have secrete less GH and produce less IGF-I,
while mRNA levels of the IGF-I receptor and GH receptor in hepatic cells are unchanged.
Oral administration of citrate can correct these levels by generating more bicarbonate, that
neutralises the pH2.
In normal cells, the expression of hexokinase is regulated by insulin. Cancer cells however
overexpress insulin-like growth factors (IGF’s) and its receptors. IGFs show a high homology
with insulin in structure, and therefore they can stimulate hexokinase, and thus glucose uptake
and metabolism. Because of the many effects of IGF, the hypothesis is that IGF may, at least
in part, be responsible for the overexpression of hexokinase activity. This enzyme stimulates
the of the first step of the glycolysis, an overproduction of pyruvate and thus the formation of
a high level of lactate13.
1.5: Relationship between lactic acidosis and hypoglycaemia
When lactic acidosis is diagnosed in a patient with non-Hodgkin lymphoma, the liver function
is assessed. If this is lower than normal, this suggests that the liver contributes to lactic
acidosis by a lower clearance of lactate.
Patients with non-Hodgkin lymphoma and lactic acidosis, sometimes also suffer from
hypoglycaemia. When plasma lactate levels are elevated, this may point to a block in the
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conversion of lactate to oxaloacetate, due to a dysfunction of the mitochondria. As can be
seen in figure 1, this means that gluconeogenesis is inhibited, leading to hypoglycaemia6.
1.6: Diagnoses of cancer by the Warburg effect
Because the Warburg effect applies to most of the leukaemia’s and lymphomas, lactic
acidosis can be used as a diagnostic marker. This method is named
18
fluorine labelled 2-
deoxyglucose positron emission tomography imaging (FDG PET imaging). FDG PET
imaging measures the phosphorylation of glucose, which is catalyzed by hexokinase. This
mirrors the rate of anaerobe glycolysis, and thus lactic acidosis7.
1.7: Treatment of patients with non-Hodgkin lymphoma and lactic acidosis
It is still unclear how to treat patients with non-Hodgkin lymphoma and lactic acidosis.
Aggressive chemotherapy would destroy the neoplastic cells that produce an excess of lactate.
Unfortunately, many patients do not respond favourably to chemotherapy. In addition to
chemotherapy, sodium bicarbonate can be administered, to neutralise the pH. However, it can
not solve the serum level of lactate. A side effect can be hyperosmolality and volume
overload, which both can lead to die of patients14.
A reason for lactic acidosis can be dysfunction of the kidneys. In patients with kidney failure
continuous veno-venous hemofiltration (CVVH) or intermittent hemodialysis is therefore
used. These methods are renal replacement therapies, what is used for acute renal failure. In
patients with lactic acidosis without lymphomas, this treatment helps very well, because it
helps eliminates the excess lactate. In malignant diseases however, lactate levels remained
unchanged510. After a few days most of the patients died4.
Lactic acidosis can be treated with insulin, what facilitates oxidation of lactate to pyruvate, by
increasing the inversion of pyruvate into acetyl-coenzyme A. However, because of a greater
source of glucose, also more lactate is formed, because of the increased availability of
glucose5.
Lactic acidosis can be caused by a thiamine deficiency. For that reason the thiamine levels in
the blood of the patients are measured. If these levels are decreased, patients are administered
with a vitamin cocktail, which contains thiamine, what will solve lactic acidosid in most of
these cases10.
Alkalinisation is used to turn the pH to its normal level. However, this treatment appeared to
be not that affective, but it may be necessary to prevent the cardiovascular system against the
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acidosis. When cardiac output is insufficient in patients with lactic acidosis, there can be an
administration of catecholamines, because responsiveness to catecholamines is decreased in
cardiac output insufficiency. If administration with catecholamines does not work, pH can be
corrected, by administration of bicarbonate. However, 10-15% is immediately converted to
CO2, what results in a decrease in pH5.
However, until the cause of lactic acidosis is not entirely clear, a good therapy cannot be
provided.
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Chapter 2: Non-Hodgkin Lymphoma (NHL)
Non-Hodgkin lymphomas (NHL) include several malignant diseases which are variations on
the Hodgkin lymphoma. NHL is a lymph proliferative disease which represents 3-5% of all
cancers registered in Europe.
The various types of disease exhibit different histological and clinical appearances. This is the
reason that NHL is difficult to diagnose.
2.1: Diagnosis
Patients with non-Hodgkin lymphoma suffer from several vague symptoms, which do not
immediately indicate a malignant disease. Patients can have several symptoms like pain,
decreased appetite, weight loss, fever, night sweats, cough and weakness. When patients are
physical examined, diagnoses of lymphadenopathy, splenopathy, larger liver and kidneys,
pleural exudate, oedema, abdominal swelling, somnolence, tachypnea and tachycardia are
done.
Lymphadenopathy can cause compression of other tissues like the ureter or spinal cord. Rapid
tumour growth in aggressive lymphomas causes severe illness14514.
Serum levels
In all patients that has been described in case reports, different blood tests are taken.
Maintenance of homeostasis in the blood is very important, and for that reason it is important
that pH in blood is 7.4. Patients with NHL however get often lactic acidosis, what results in a
lower pH. Lactic acidosis is therefore determined when pH is lower than 7.35 and when the
concentration of lactate is higher than 5 mmol/L143.
In different studies it appeared indeed that patients got a lower pH, with variations from 7.03
till 7.39. Lactate concentrations differed from each other in big variations, with outliers of
46.8 mmol/L4. Another way to confirm acidosis is to measure the level of bicarbonate. If this
level is too low, it is another sign of acidosis5.
To diagnose the disease the leukocytes are counted. Besides of this examination, also the
morphology of the cells can be investigated. These results can give the diagnosis of having
leukaemia and/or lymphomas5.
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Because some people also appeared to have hypoglycaemia, serum levels of glucose are
measured. Hypoglycaemia is the situation in the body when serum glucose is lower than 40
mg/dL5.
In all patients the concentrations IGF and IGFBP were measured. IGF-I, IGF-II and IGFBP-3
levels were low, while levels of IGFBP-1 and IGFBP-2 tended to be high. Also levels of
TNF-α were measured, and an increased level of lactate led to elevated TNF-α levels5.
In these patients different methods are used to measure levels in the blood. Radioimmunoassay and acid-alcohol extraction are used to measure levels of IGF-I and IGF-II.
Levels of IGFBP are measured with immunochemolucent assay5.
In one study also the level of thiamine is measured, because lack of this vitamin can have
lactic acidosis as a result. Three patients had a thiamine deficiency, and after administration
with thiamine, the lactic acidosis decreased10.
Physical examination
Of all patients that are studied, most of the patients are examined with computed tomography
(CT) scan, to see whether organs were abnormal or not. As already said, an abdominal
swelling can be seen, just as splenomegaly and lymphadenopathy (fig.4)4.
Also an increased size in liver can be seen, 81% of all the patients appeared to have liver
involvement4. However, patients have normal liver parenchyma, what suggests that the liver
produces tumour cells, which produce a lot of lactate. Besides liver failure, also the kidneys
are involved145.
Fig. 4 4 Computed tomography (CT) scan of the abdomen. L; liver, S; spleen, N; lymph node. All these organs
are increased in size.
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The diagnosis for a malignant disease can be from biopsies on bone marrow and lung. For
example in one study, exudate from the lungs showed malignant cells, while bone marrow
biopsy showed high-grade, small noncleaved cells, what Burkitt’s lymphoma suggested. This
diagnosis was confirmed after chromosomal analysis14.
2.2: Pathology
NHL is a disease that is caused by a dysfunction of the B-lymphocytes. In the human body, Blymphocytes differentiate in the central lymphoid tissue (bone marrow and thymus) and then
migrate to peripheral lymphoid tissues. Differentiation of the B-lymphocytes is characterized
by cytological changes and alterations in homing mechanisms. This is caused by changes in
gene expression, especially involving the proto-oncogenes and tumour suppressor genes.
Chromosomal translocations cause deregulated expression of proto-oncogenes, and these
translocations are represented in each tumour, except in T-cell anaplastic large cell lymphoma
and mucosa-associated lymphoma tissue (MALT)1.
2.3: Different types and their treatment
The type of treatment depends on the type of non-Hodgkin lymphoma. There is not yet a good
treatment for patients with NHL, lactic acidosis and hypoglycaemia, because it is not known
what the cause is of death. This can be seen of the high death rate.
Non-aggressive lymphomas
Follicular lymphoma
This type of lymphoma is the most abundant form of non-Hodgkin lymphoma. The Bcl-2
gene is involved, which is a proto-oncogen that encodes pro-apoptotic proteins and antiapoptotic proteins.
When this lymphoma is localised, it can be treated with radiotherapy. On the average, in 98%
of cases this leads to remission, and in 47% of cases in 10 years survival. In most patients the
lymphomas are disseminated, and radiotherapy is useless. For most patients this disease is
incurable, and medicines can only delay the disease.
Rituximab is a monoclonal antibody IgG that binds to CD20, an antigen on the surface of a Bcell. Binding of the antibody causes cytotoxic effects, complement-mediated, antibodydependent cytotoxicity in the B-cell and it induces apoptosis. In combination with standard
chemotherapy it has the strongest effect1.
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Transformed non-aggressive lymphoma
Lymphomas can transform from non-aggressive lymphomas to aggressive lymphomas and
therefore high-grade malignancies. For this reason, the treatment is the same as for high-grade
lymphomas, but 40% of the patients die from this disease1.
Chronic small lymphocytic lymphoma
This lymphoma is chronic but slowly progressive, which is mostly present in adults older than
60 years. The disease is characterised by lymphadenopathy. Pax5 is the proto-oncogene which
is involved, a transcription factor that regulates the proliferation and differentiation of B
cells1.
Mantle-cell lymphoma
Proto-oncogenes Bcl-1 and cyclin D1 are involved in this type lymphoma, which are cellcycle regulators. This type of non-aggressive lymphoma is the most aggressive, and median
survival is about 3 years. Treatments that are used are immune therapy and chemotherapy
with antibodies against CD20, with or without radio labelling (fig.5)1.
Fig. 5 1 Therapy in non-Hodgkin lymphoma: A. an antibody against CD20, a specific antigen for B-cells. B. An
antibody, labelled with a radiolabel.
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Melanie van Berkum, April-July 2011
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Aggressive lymphoma
Diffuse large B-cell lymphoma
The major proto-oncogene that is involved is Bcl-6, a repressor of the transcription.
Radiotherapy is not useful, and therefore patients receive chemotherapy, primarily Rituximab
to CHOP (a combination of cyclophosphamide, vincristine, doxorubicin and prednisolone),
followed by radiotherapy1.
Burkitt’s lymphoma
This type of lymphoma occurs most frequently in childhood and c-myc is involved, a
transcription factor that regulates proliferation and growth1. It is a tumour which is comprised
of small noncleaved cells14.
A complication of this type is an increased concentration of lactate dehydrogenase in the
blood. Patients will receive chemotherapy
Peripheral T-cell lymphoma
Unlike the other types, this type is caused by dysfunction of the T-cells. It is caused by the
HTLV-1 virus, and it is a very aggressive form1.
Extranodal lymphoma
Extranodal lymphomas can reside in the central nervous system. This type of lymphoma, is
very aggressive with a low 5-years survival rate. Another extranodal lymphoma can be
located in the testis, especially in men older than 60 years.
Gastric lymphomas of MALT type is a kind of lymphomas that is related to infection with
Helicobacter pylori. Use of antibiotics decreases the size of lymphomas in lung and thyroid
gland. The survival of patients is high1.
When patients with NHL are admitted to the hospital, they often get chemotherapy, to reduce
the size of the tumours, and therefore lactic acidosis will disappear. Lactate concentration
decreased indeed after chemotherapy, but after a while, lymphomas recurred, and lactate
levels went back to the normal. Hypoglycaemia can be corrected with administration with
glucose, to increase the concentration of glucose in the blood5.
To summarise, the health of patients will improve after chemotherapy, but all patients
eventually will die because of the combination of lactic acidosis and NHL5.
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Chapter 3: The IGF-system
3.1: The growth hormone-releasing hormone-growth hormone IGF-axis
The hypothalamus produces somatostatin and growth hormone-releasing hormone (GHRH).
GHRH stimulates the anterior pituitary gland through the GHRH-receptor to produce growth
hormone (GH), whereas somatostatin inhibits this process. After binding to the GH receptor,
GH induces the production of IGF-I in many organs (figure 6). Especially the liver contributes
(for about 80 %) to the circulating IGF-I pool. IGF-I may act also in a paracrine or autocrine
manner. In contrast, IGF-II is not regulated directly by GH.IGF-I and IGF-II show a high
degree of homology with respect to their amino acid sequences. Mature IGF-I (7.65 kD) and
IGF-II(7.47 kD) contain 70 amino acids (7,65 kD, and 67 amino acids, respectively)15,16,17.
IGFs show also a great similarity with insulin, i.e. 48% of the amino acids are identical, with
the same disulfide bonds. However, IGFs still contain the C-peptide which is cleaved from
pro-insulin, to form insulin. Under normal circumstances, in the circulation of adult humans
the concentration of IGF-II exceeds that of IGF-I more than 3-fold16.
Fig. 6
18
The GH-GHRH-IGF-axis. The hypothalamus excretes GHRH that will go to the pituitary, what
produces GH. GH will be transported to the liver, where it will produce IGF-I. GH and IGF-I both have negative
feedback to the pituitary and the hypothalamus, like hyperglycaemia and cortisol. Stress, amino acids,
hypoglycaemia, oestrogen and testosterone will stimulate the hypothalamus and the pituitary.
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3.2: IGF-I and IGF-II receptors
IGF-I receptor
The structure of the IGF-I receptor (IGF-IR) (180 kD) resembles that of the insulin receptor
(IR), which is expressed in two isoforms (IR-A and IR-B) differing by 12 amino acids due to
the alternative splicing of exon 11. Both IGF-IR and IR are tetramers consisting of two α and
two β subunits, showing 60 % amino acid homology. Another similarity is the presence of a
tyrosine kinase domain, which is 84 % identical. The IGF-I and IRs have a different
cytoplasmic C-terminal domain. The IGF-IR stimulates DNA synthesis more effectively than
IRs, but various other effects of the receptors are similar16.
The two α subunits of the IGF-IR contain the extracellular IGF binding sites. These subunits
are covalently linked to two β subunits that stick through the plasma membrane. The
intracellular parts of the β subunits contain tyrosine kinase domains. After binding of IGF-I, a
conformational change takes place in the receptor, and the intracellular tyrosine kinase
domain autophosphorylates specific tyrosines on effector proteins. These proteins can activate
different signal transduction cascades which are important in stimulating cell proliferation and
survival (figure 7)16.
Not only IGF-I, but also IGF-II and insulin can bind to IGF-IR. However, IGF-II binds with a
six fold lower affinity than IGF-I. The affinity of IGF-IR for insulin is 100-fold lower than
that for IGF-I16. Both IGFs also bind to IR-B, albeit with a much lower affinity than insulin.
IGF-II binds with high affinity to the IR-A isoform, with an affinity close to that of insulin.
Moreover, IGF-II binds to IR-A with an affinity equal to that of IGF-II binding to the IGF-IR. Activation of IR-A by insulin leads primarily to metabolic effects, whereas activation of
IR-A by IGF-II leads primarily to mitogenic effects19.
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Fig. 7
20
The different pathways that are triggered by the binding of insulin and IGFs to their receptors.
Phosphorylation of the receptors results in phosphorylation of other proteins that triggers important pathways in
the cell, like transcription, cell survival, protein synthesis, glycogen synthesis and glucose uptake.
IGF-II receptor
The IGF-II receptor only binds IGF-II, with high affinity, but not to insulin. It differs from the
IGF-IR and IRs in that it lacks in tyrosine kinase activity. It competes with IGF-IR and IR-A
for IGF-II binding, and therefore prevents the proliferative and anti-apoptotic effects of IGFII. The receptor is identical to the mannose-6-phosphate receptor, and for this reason this
receptor is called the M6/IGF-IIR16. It merely serves mainly as a scavenger receptor21.
IGF-IR-IR hybrids
Because of the structural similarity between IGF-IR and IRs hybrids, i.e. αβ hemireceptors,
of these receptors may be formed in cells which express both types of receptors. These hybrid
receptors bind to IGF-I and IGF-II with high affinity, but with low affinity to insulin.
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The physiological meaning of the existence of these receptors is still unclear. It has been
shown that this hybrid receptor can interact with cell-cell adhesion complexes and estrogen
receptor signalling16.
3.3: Effects of IGF-I and IGF-II
IGF-I
Overall, IGF-I exerts proliferative effects, induces differentiation, and prevents apoptosis.
Hence, IGF-I mainly plays a role in growth-related processes. The GH and IGF-I systems are
major regulators of the metabolism of the bones. These hormones stimulate the proliferation
and differentiation in the skeletal growth centre.
Also in other organs, like the mammary glands they induce mammary gland cell proliferation,
and have a role in pregnancy and growth. In the muscles it induces growth, but it also induces
hypertrophy and it helps to regenerate skeletal muscles. In the heart IGF-I is protective against
stroke and other cardiovascular diseases, since it acts as an anti-apoptotic factor on vascular
cells, which results in the maintenance of vascular endothelium and smooth muscles. In
neuronal cells, IGF-I is an factor for the survival of the cells, and it is protective against
diseases like Alzheimer and Huntington’s disease.
On the other hand, a higher expression of IGF-I or IGF-IR can also be a risk factor for
different types of cancers.
IGF-I has metabolic effects, such as stimulating protein synthesis and preventing proteolysis.
In addition, it has insulin-like effects, what causes a lower level of glucose in the blood, by
stimulating glucose transport16.
IGF-II
IGF-II plays a role in embryonic and foetal growth. In addition, many types of cancerous cells
over express IGF-II, stimulating neoplastic growth in a paracrine or autocrine fashion. In
vitro, the biological effects of IGF-II are merely similar to those of IGF-I, and are mediated
by either IGF-I or IR-A. The interaction of IGF-II with these receptors predominantly lead to
mitogenic effects. In contrast, the low-affinity binding of IGF-II to IR-B results in insulin-like
effects22.
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Fig. 8
21
GH binding to the GH receptor complex. After binding, JAK2 is recruited what activates Ras/MAPK
and STAT5b (signal transducer and activator of transcription type 5b). This is a transcription factor for ALS,
IGFBP-3 and IGF-I, what forms a 150 kDa complex. When ALS-gene is mutated in the liver, levels of IGFBP-3
and IGF-I decreases. In extrahepatic tissues, IGFBP-3 and IGF-I are normally expressed and act in autocrine or
paracrine mechanisms.
3.4: IGF-binding proteins (IGFBP) and the acid labile subunit (ALS)
By far most of the IGFs in the circulation, i.e. under normal circumstances > 99 %, are firmly
bound to IGF-binding proteins (IGFBPs), the affinities being in the same order of magnitude
as that for the IGF-IR. At present, six different IGFBPs have been identified. The major part
of the serum IGFs (about 70 %) is sequestered in a ternary 150 kDa complex, consisting of
the acid labile subunit (ALS) and the most abundant IGFBP, IGFBP-3. This complex is not
able to pass the capillary membrane and prolongs the half life of serum IGFs considerable, i.e.
up to 15 hours when compared to the free IGFs which have a short half life time of only 10
minutes. The remaining part of the IGFs is present as binary complexes (40-60 kD) with the
various IGFBP with a half life time of about 30 minutes. The function of IGFBPs is to
regulate the bioavailability of IGFs. In addition, several IGFBPs have been shown to possess
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intrinsic IGF-independent biological effects23. The various IGFBPs and ALS are briefly
discussed below.
IGFBP-1
The first IGFBP was discovered in 1984 and named IGFBP-1. Its structure is maintained by
disulfide bonds, and in this state, at non-reducing conditions when no disulfide bonds are
broken, this protein has a mass of 32-34 kDa, with 234 amino acids. The protein has O-linked
glycosylation sites, and can be phosphorylated at the serine residues. This phosphorylation
can regulate the bioactivity of IGF-I.
The IGF-binding domain consists of a cysteine-rich region at the C-terminus, what is required
for binding of IGF. Nearby the C-terminus also an Arg-Gly-Asp (RGD) sequence appears, a
sequence that is mostly present in cell matrix adhesion proteins. It is recognized by integrin
receptors at the cell surface, what probably facilitates the transport of IGF-I to its receptor.
Together with the negative charge, the Pro, Glu, Ser and Thr (PEST) amino acids suggests a
short half-time, because this is also seen in other proteins with a PEST region. Hepatic
IGFBP-1mRNA expression is inversely regulated by insulin, i.e. reduced by increasing levels
of insulin.
Levels of IGFBP-1 are high in children, but in puberty this level is decreased and in a steady
state. It is elevated during fasting, in diabetes and during pregnancy. After therapy with
insulin the levels of IGFBP-1 decreased. IGFBP-1 is mostly present in the liver24.
IGFBP-2
This IGFBP contains 289 amino acids in mature protein, and is in non reducing conditions 36
kDa. Like IGFBP-1, IGFBP-2 contains a RGD sequence near to the C-terminus, for its
association with integrin cell surface receptors. Both the N-terminus as the C-terminus have
affinity for both IGFs, however IGFBP-2 has a preference for IGF-II over IGF-I.
Concentrations of IGFBP-2 are the highest in the liver, but are also found in the stomach,
kidney, lung and brain24.
IGFBP-3
IGFBP-3 is the most abundant binding protein in the human serum, being mostly associated
with an IGF and ALS molecule to form a complex of 150 kDa. Under non-reducing
conditions IGFBP-3 can be 53 and 47 kDa, with 264 amino acids. There are three N-linked
glycosylation sites. The C-terminus is important for the binding with IGF and ALS, while the
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N-terminus is also needed for binding with IGF-I. In pregnant women a proteolytic enzyme is
reported, specific for IGFBP-3. This leads to fragments of 26 kDa and fragment of 17-22 kDa.
IGFBP-3 levels are low at birth, high in puberty and low in adults. IGFBP-3 expression is
regulated by GH. In patients with GH deficiency, serum levels of IGFBP-3 are reduced. In
patients with acromegaly, levels of GH and IGFBP-3 are both increased. IGFBP-3 is
regulated too by IGF-I, insulin and mitogens like vasopressin and EGF24.
Both in solution and at the cellular level, IGFBP-3 is considered to play an important role in
the regulation of the interaction between IGF-I and the IGF-IR. IGFBP-3 has been shown to
interact with various extracellular matrix components, through its heparin binding domain.
Cell associated IGFBP-3 competes with the IGF-IR for IGF-I binding, and hence inhibiting
IGF-I action. It has been postulated that the extent of IGF-I binding to IGFBP-3 at the cell
surface is, at least in part, controlled by local IGFBP-3 protease activity25.
IGFBP-4
IGFBP-4 contains one N-linked glycosylation site, and is primarily synthesised in the liver. It
exists as a non-glycosylated protein (24 kDa) and a glycosylated protein (28 kDa)24. It inhibits
IGF-I by inhibiting the interaction of IGF-I to the receptor. This will inhibit cell
proliferation2627.
IGFBP-5
This binding protein is composed of 252 amino acids without N-linked glycosylation sites. It
can be 29 kDa, but after purification by a protease the protein is 24 kDa24. As IGFBP-3,
IGFBP-5 may form a large complex with an IGF and ALS.
IGFBP-6
This binding protein has a higher affinity for IGF-II than for IGF-I, by the unique structure of
its N-terminus, and it has one potential N-linked glycosylation site24.
ALS
ALS is a leucine-rich glycoprotein of 85 kDa that is produced by the liver and then is secreted
into the circulation. Its synthesis is stimulated by GH. It belongs to a family of proteins that
are involved in protein-protein interactions, because of its negative charged residues and
electronegative surfaces. As emphasized previously, the major function of ALS is to maintain
circulating IGFs into 150 kDa complexes with IGF-I (or IGF-II) and IGFBP-3. In this
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manner, IGFs are protected from degradation and proteolysis28293021. Not only the half-life’s
of IGFs are prolonged, but also that of IGFBP-3, since the passage of IGFBP-3 from the
intravascular to the extra vascular space is decreased28.
The lack of ALS has different effects on the GH-IGF system. Circulating levels of IGF-I,
IGF-II, and IGFBP-3 decrease considerably, because of the instability of the proteins, while
GH levels are elevated21.
It is not clear yet whether ALS has other biological functions. It has been suggested that ALS
is involved in the regulation of fat and carbohydrate metabolism. ALS knockout mouse
models are growth deficient, and show decreased serum levels of IGF-I and IGFBP-3, due to
increased turnover rates. These mice also show a faster clearance rate of glucose, and normal
levels glucose are measured during fasting21.
In spite of the very low levels of IGFs and IGFBP-3, patients with ALS deficiency exhibit
only relatively mild growth retardation and delayed puberty. Presumably, part of the growth
can be maintained by locally produced IGF-I. It is an autosomal recessive inherited disease,
and there are different types of mutation possible31.
Another result of ALS deficiency in humans is insulin insensitivity, diagnosed by low levels
of IGFBP-I, a marker of insulin sensitivity. It is not clear how ALS deficiency causes insulin
insensitivity. It has been hypothesized that the very low levels of IGF-I in ALS deficient
patients lead to an increase of GH secretion. The metabolic effects of GH counteract those of
insulin, leading to insulin resistance21.
3.5: The IGF system under pathological conditions
The IGF-system is involved in various growth disorders, e.g. GH deficiency, acromegaly, and
malnutrition. In addition both IGFs as well as several IGFBPs are considered to play an
important role in tumorigenesis. These aspects, however, are beyond the scope of this thesis.
In this thesis, only the relationship between the IGF-system and acidosis, non-islet-cell
tumour-induced hypoglycaemia, and leukaemia (with a focus on NHL), respectively, will be
reviewed.
Acidosis
In NHL lactic acidosis causes a change in the IGF system, a change that is also seen in other
types of metabolic acidosis. Acidosis causes protein degradation, what results in growth
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failure. This can be the result of decreased food intake, acidosis in blood, and the abnormality
of the GH-IGF axis. However, the mechanisms are not known yet3212.
Patients with carbonic anhydrase II deficiency (CAD) appeared to have metabolic acidosis in
the renal tubuli of the kidney. These patients have a decreased secretion of GH, and show
growth retardation, as a result of a decreased synthesis IGF-I in the liver. Administration of
GH in mice with mild metabolic acidosis resulted in an increase in the serum IGF-I level.
Administration of GH, however, is not effective in severe types of metabolic acidosis3233.
Studies on the mRNA level revealed that expression of both the GH receptor and IGF-I genes
had decreased, even though the transcriptional machinery of IGF-I was still intact, based on
the fact of the increase in IGF-I after GH treatment in mild types of metabolic acidosis32.
Changes in pH results in an aberrant function of different proteins. IGF-I, IGFBP-3 and ALS
normally forms a complex to increase the half-life of IGF-I. Acidosis changes the affinity of
these molecules, what results in an increase in free IGF-I, as is mentioned below15.
However, acidosis increases the expression of IGFBP-2 and IGFBP-4, two binding proteins
which inhibit IGF-I action26.
Regulation of IGF-I bioactivity by IGFBP-3
Both in solution and at the cellular level, IGFBP-3 is considered to play an important role in
the regulation of the interaction between IGF-I and the IGF-IR. IGFBP-3 has been shown to
interact with various extracellular matrix components, through its heparin binding domain.
Cell associated IGFBP-3 competes with the IGF-IR for IGF-I binding, and hence inhibiting
IGF-I action. It has been postulated that the extent of IGF-I binding to IGFBP-3 at the cell
surface is, at least in part, controlled by local IGFBP-3 protease activity25.
In vitro
experiments with bovine mammary epithelial cells demonstrated that acidification of the
culture medium leads to enhanced binding of IGF-I at the cell surface. This appeared to be
facilitated by an increased cell surface association of IGFBP-3 at reduced pH. It was
hypothesized that the observed increment of cellular IGF-I binding at lower pH values was
due to increased binding of IGF-I to IGFBP-3 and not to IGF-IR. This increase in affinity may
be caused by protonation of ionisable groups on the two molecules. The electrostatic
interaction will be changed, and salt bridges or H-bonded are formed23. In addition, in the
presence of exogenous IGFBP-3, at acidic pH cell proliferation was reduced and an increased
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internalization and nuclear association of IGF-I was observed. This would suggest that IGF-I
bound to IGFBP-3 is localized away from IGF-IR, explaining the decreased mitogenic effects
of IGF-I. Hence, this would mean that an acidic microenvironment, as in the case of highly
metabolically active tumour cells, could have a favourable, i.e. inhibiting, effect of IGFs on
tumour growth2334.
On the other hand, using a variety of different cell lines, Conover et al.
35
showed that
radiolabeled IGFBP-3 remained virtually intact when the cells were cultured at neutral pH.
Acidification of the culture medium ( pH < 5.5), however, lead to significant hydrolysis of
IGFBP-3. It was found that this was caused by an activation of the aspartic proteinase
cathepsin D that has an acidic pH optimum. Addition of an antibody to cathepsin D reduced
IGFBP-3 proteolysis. It is known that proteolytic fragments of IGFBP-3 lack high affinity
binding of IGF-I, allowing the sequestered IGF-I to bind to IGF-IR.
It may well be that an increased local proteolysis of IGFBP-3 counteracts, and overrules, the
inhibitory effect of the originally intact IGFBP-3 on IGF-I binding to the IGF-IR. Hence, the
net effect of local acidification would be an increased proliferation of tumour cells which is in
agreement with the poor prognosis of hematologic malignancies complicated by lactic
acidosis5.
Leukemia
In patients with NHL the levels of the molecules of the IGF-system are changed. IGF-I, IGFII and IGFBP-3 levels are decreased, while IGFBP-1 and IGFBP-2 levels are increased.
Levels of the precursor of IGF-II, pro-IGF-II, are not changed, but this molecule changed in
structure and is transformed into a high-molecular-weight IGF-II, big IGF-II5.
When circulating levels of IGFBP-2 are increased, this may lead to an increase in the
proliferation of the peripheral blood mononuclear cells (PB MNCs). Elevated levels of
IGFBP-2 therefore are considered to be related with malignant diseases36.
When people are GH deficient or insensitive, levels of ALS, IGF en their binding proteins are
strongly reduced23.
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Non-islet-cell tumour-induced hypoglycaemia (NICTH)
Data on the exact incidence and prevalence of NICTH are lacking. Probably many cases are
not diagnosed. NICTH occurs mainly in patients with a solid tumour of mesenchymal and
epithelial origin. But, although rarely, it has also been encountered in patients with tumours of
haematopoietic origin22.
NICTH is a form of hypoglycaemia occasionally present in patients with malignant diseases.
Hypoglycaemia takes place when patients are fasting: the liver does not produce enough
glucose because of inhibition of the gluconeogenesis and glycogenolysis. Lipolysis is also
decreased, and levels of free fatty acids are decreased. In addition, due to the low glucose
levels insulin secretion is inhibited. The lack of glucose is also the result of the high-use of
glucose of the tumour. Hypoglycaemia is a severe symptom because it can result in sweating,
lethargy, somnolence and a decreased motor activity, what can lead in the worst case scenario
to coma22.
In subjects with NICTH levels of IGF-I are decreased, levels of IGF-II can be either normal,
decreased or increased. Levels of IGFBP-2 are mostly increased22.
NICTH is considered to be caused by an
aberrant functioning of the IGF-system. The
secretion of incompletely processed precursors of IGF-II (‘big’-IGF-II) by the tumour cells is
induced. The high insulin-like activity of ‘big’-IGF-II leads to recurrent episodes of fasting
hypoglycaemia. This in turn induces profound changes in the circulating levels of insulin,
GH, IGF-I and several IGF-binding proteins,
Circulating big IGF-II competes with IGF-I and mature IGF-II for binding with IGFBP3.
However, big IGF-II-IGFBP-3 complexes cannot form 150 kD complexes with ALS. As a
consequence, increasing amounts of IGFs and big IGF-II will either form binary complexes
with IGFBPs or remain unbound37. In this way, the bioavailability of IGFs for the tissue
compartments will increase. Elevated levels of free IGFs, would suppress the secretion of GH
and insulin. When insulin is suppressed, IRs will be upregulated to increase the sensitivity to
both insulin and IGFs. Suppression of GH also causes a decreased production of IGF-I,
IGFBP-3, and ALS, but an increased production of IGFBP-2, which aggravates the impaired
formation of the 150 kDa complex. Together with the increased insulin sensitivity, an
increased consumption of glucose is the result, leading to hypoglycaemia37.
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Fig. 9 22 Hypoglycaemia in patients with NICTH The tumour uses high amounts of glucose, the first cause of
hypoglycaemia. The tumour cells increases production of IGFBP-1, -2, -4, and -6, and also ‘big-IGF-II. The liver
produces however less IGF-I, IGFBP-3 and -5, and ALS. This results in less formation of 150kDa compexes of
IGF-I, IGFBP-3 and ALS, and an increased formation of IGF-II and IGFBP-3, complexes of 40-50kDa. More
free IGF-I and IGF-II is available, what results in an increased glucose consumption in the adipose tissue and
muscles, while less glucose is formed in the liver and the adipose tissue. All these processes will lead to
hypoglycaemia.
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Chapter 4: The IGF-system, lactic acidosis and leukaemia (NHL): case
reports
Lactic acidosis, i.e. when the pH of patient’s serum is lower than 7.37, with a lactate level
greater than 2 mmol/L, is a complication of especially leukemia’s and lymphomas, including
NHL. In some cases patients may also suffer from hypoglycemia (i.e. serum glucose level <
40 mg/dl). Until 2001, more than 28 cases of lactic acidosis associated with lymphoma have
been reported in both adults and children, of which nine cases also reported hypoglycaemia.
Four of these patients had Hodgkin disease, whereas 24 had NHL. NHL in combination with
lactic acidosis has a poor prognosis. Seventy-three % of the patients die within a month. In
addition, 25 cases with lactic acidosis associated with leukaemia are known, 11 of these
patients also showing signs of hypoglycaemia14.
Although many factors may contribute to the high rate of glycolysis that causes lactic
acidosis, the overexpression of rate limiting glycolytic enzymes seems to be a major feature.
Several studies have shown the involvement of a high expression of hexokinase II, an enzyme
being bound to the mitochondria. The expression of hexokinase II is controlled by insulin.
However, also IGFs play an important role in the regulation of hexokinase activity through
activation of IGF-IR which is often overexpressed by tumour cells.
Serum levels of IGFs, IGFBP-1, -2, and 3 have been determined in only a few cases of
lymphoma with lactic acidosis. In all cases, circulating levels of IGF-I, IGF-II, and IGFBP-3
were either below or in the lower part of the respective normative ranges. Serum levels of
IGFBP-2 were elevated in all three cases investigated, whereas IGFBP-1 appeared to be
elevated in 2 patients. It has been found that malignant lymphocytes can produce excessive
amounts of IGFBP-2. This may explain the enhanced levels of this IGFBP in the cases as
referred to above.
During treatment, the levels of IGFs and IGFBPs investigated tended to normalize. Possibly,
the decreased serum levels of IGF-I and IGFBP-3 could be attributed to acidosis-induced GH
resistance leading to diminished hepatic production of IGF-I and IGFBP-3, as observed in
acidotic rats38. Another explanation was provided by Sillos et al. They noticed that the
changes in serum levels of IGFs and IGFBPs in the patients with lactic acidosis and
lymphoma resemble the picture seen in patients with NICTH. Hence, it was hypothesized that
tumour cells may produce big IGF-II subsequently leading to suppression of the GH-IGFaxis. Such a phenomenon would also explain the episodes of hypoglycemia which may occur
in part of the patients with lymphoma and lactic acidoisis. However, levels of big IGF-II and
free IGFs have not been determined.
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Chapter 5: Various components of the IGF-system in a patient
with non-Hodgkin lymphoma and lactic acidosis
M. van Berkum, 2011
Summary
In the present study, various components of the insulin-like growth factor (IGF)-system,
i.e. IGF-I, IGF-II, IGF-binding protein (IGFBP)-1, -2,-3, -4, and -6, acid labile subunit
(ALS), 150 kD complex formation, and IGFBP-3 proteolysis, were investigated in serum
from a patient who was diagnosed with non-Hodgkin lymphoma and lactic acidosis.
Serum samples taken both before treatment and during treatment were available for
analysis. The patient was treated successfully with Rituximab, a monoclonal antibody
being combined with chemotherapy, which also resulted in the abolishment of acidosis.
Before treatment of NHL and in the presence of acidosis, the concentration of IGF-I in
patient’s serum was in the lower part of the normal range. Circulating levels of IGF-II,
IGFBP-4, and IGFBP-6 were in the upper part of the normal range, whereas that of
IGFBP-2 was clearly elevated. IGF-II in patient’s serum was in the lower part and the
upper part of the normal ranges, respectively. The levels of IGFBP-3 were between –
and + 1 SDS. Serum pro-IGF-II[68-88] was within the normal range, strongly suggesting
that the tumour cells did not produce large amounts of this compound. During
treatment, serum levels of IGF-I, IGF-II, and IGFBP-3, had increased markedly
exceeding the respective normative ranges. Serum levels of IGFBP-1, IGFBP-2, IGFBP4, and IGFBP-6 had decreased significantly, when compared to pre-treatment values.
The same was found for pro-IGF-II[68-88] which declined below – 2 SDS. The levels of
ALS in patient’s sera as determined by ELISA were within the normal range. Moreover,
no abnormalities of the ALS were observed after Western blotting of patient’s sera.
Interestingly, 150 kD complex formation in pre-treatment serum of the patient was
disturbed, as revealed by S200 column chromatography after incubation of the serum
with 125I-IGF-I. The column profile of sera tended to normalize during treatment. It
may well be that at lower pH values the interaction of ALS with the binary complexes is
hampered. As a consequence, a relatively higher proportion of IGFs is present in binary
complexes that may allow an increased access of IGFs to target cells including the
malignant ones. IGFs exert insulin-like effects, and also stimulate the expression of
hexokinase, what results in further deregulation of the glycolysis and aggravate acidosis.
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
34
Introduction
applied
in
chemotherapy,
Insulin-like growth factors (IGFs) and the
designed
IGF-I receptor (IGF-IR) play an important
Lymphomas
role in the proliferation and the metabolism
Rituximab, a monoclonal antibody which is
of
malignant
malignancies
for
aggressive
especially
(NHL).
It
Non-Hodgkin
consists
of
cells.
Hematologic
added, to the standard combination called
as
non-Hodgkin
CHOP.
such
CHOP
consists
of
lymphoma are sometimes associated with
cyclophosphamide, doxorubicin, vincristine,
lactic acidosis. The possible involvement of
and prednisolone. The first three drugs of
the IGF-system in the pathogenesis of this
the CHOP chemotherapy regimen are
disease is poorly understood.
usually given i.v. on one single day, while
In the present study, various components
prednisolone is taken orally for five days.
of the IGF-system, i.e. IGF-I, IGF-II, IGF-
Each cycle is repeated every 3 weeks for 6-8
binding protein (IGFBP)-1, -2,-3, -4, and -6,
cycles.
acid labile subunit (ALS), 150 kD complex
formation, and IGFBP-3 proteolysis, were
Patient’s serum samples
investigated in serum from a patient who
was
diagnosed
with
lymphoma and lactic
non-Hodgkin
acidosis.
Three blood samples were taken, one before
Serum
start of treatment, i.e. during lactic acidosis,
samples taken both before treatment and
and two samples during R-CHOP therapy
during treatment were available for analysis.
(see below), at 22 and 25 days, respectively.
Materials and methods
Determination of serum levels of GH, IGF-
Case presentation
I, total IGF-II, pro-IGF-II[E68-88], and
IGFBP-3
A 79- years old male patient was diagnosed
with a diffuse large B-cell lymphoma, an
aggressive
form
of
non-
Hodgkin
lymphoma. The patient presented with lactic
acidosis (i.e. serum lactate level >5 meq/L
and pH<7.35). After some delay, he was
treated successfully with R-CHOP and
acidosis had abolished. R-CHOP is the
These parameters had been determined
previously by technicians of the Laboratory
of Endocrinology. In brief:
IGF-I was measured using an immunometric
technique on an Immulite 1000 Analyzer
(Siemens Medical Solutions Diagnostics,
Los Angeles, USA).
abbreviated name of a combination of drugs
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
35
The lower limit of detection was 12 ng/mL
sample, 50 μl antiserum BP6 (1:125 diluted)
and inter-assay variation was
and 50 μl 125I-hIGFBP-6 tracer.
<7,5% at 50-420 ng/mL (n=200)
After overnight incubation at 4 ºC, 100 μl
Total IGF-II levels were determined in Sep-
Saccel
Pak C18 extracts of plasma by RIA, as
incubation of half an hour at room
described previously (1,2) For the present
temperature, 1000 μl distilled water was
study, native hIGF-II isolated from Cohn
added, and the tubes were centrifuged. The
fraction IV of human plasma was used as a
pellets were counted in a γ-counter.
anti-rabbit
was
added.
After
standard and for 125 I labeling. At a mean
level of 505 ng/mL (n=12) the intra- and
IGFBP-4 RIA
inter assay coefficients of variation were 6.7
and 8.8 %, respectively. The sensitivity of
the assay was 0.09 ng/mL. The RIA was
calibrated against the WHO reference
preparation of recombinant (r) hIGF-II (3).
IGFBP-3 was determined by specific RIA,
GH was determined by the Immulite hGH
from
Siemens
Medical
new standard preparation of recombinant
hIGFBP-4 had to be used. The assay was
performed five times in order to compare
the data obtained with this new standard
preparation with those obtained with the one
as described previously39.
kit
This assay had to be recalibrated since a
Solutions
Diagnostics (Los Angeles, USA) on a
Immulite 1000 Analyzer.
that has been used previously. This factor
was 3.03. The standard (1μg/ml) was diluted
six times, at which the last standard had a
concentration of 1.56 ng/ml. The assay
buffer was composed as described earlier27.
IGFBP-6 RIA
The incubation mixture of the standards
consisted of 200 μl buffer, 100 μl standard,
The levels IGFBP-6 in the serum samples
50 μl antiserum BP4 and 50 μl tracer BP6.
was determined as described previously39. In
The incubation mixture of the samples and
brief: Recombinant hIGFBP-6 standard (160
controls consisted of 250 μl buffer, 50 μl
ng/ml) is diluted serially seven times, the
sample, 50 μl antiserum BP4 and 50 μl
last standard has a concentration of 1.25
hIGFBP-4 tracer.
125
I-
ng/ml. The assay buffer (IGFBP-3 buffer)
was composed of 0∙2% BSA, 50 mM
sodium phosphate (pH 7∙4) and 0∙05 (w/v)
Tween-20. The incubation mixture consisted
of 250 μl IGFBP-3 buffer, 25 μl standard or
After
overnight
incubation
at
room
temperature, 100 μl Saccel anti-rabbit was
added. After incubation of half an hour at
room temperature, 1000 μl distilled water
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
36
was added, which were centrifuged. The
Analysis of IGFBP-3 and ALS by Western
pellets were counted in a γ-counter.
immunoblotting
ALS ELISA
Serum (3 μl) was diluted in 20 times
Loading Buffer. With sodium dodecyl
Serum levels of ALS were determined by
sandwich
ELISA
using
the
kit
of
Mediagnost (Reutlingen, Germany) The
ALS calibtation curve was fitted by a 5
degrees polynome algorithm.
sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) the proteins are separated,
based on size. Proteins were transferred to
Immobilon membranes, with transfer buffer,
composed of 25 mM Tris, 192 mM glycin
and 20% methanol. After washing, the
S200 column chromatography
immunoblots were incubated with Tris
Buffered Saline (TBS-buffer) with 0∙2%
The different molecular-size classes of
endogenous IGF-I (i.e. 150 kDa, 40–50 kDa,
and unbound, free form) in plasma were
determined by neutral gel filtration through
a 1.6 x 60 cm Superdex 200 Hiload column .
Prior to column chromatography, each
serum sample (250 μl) was incubated with
100 μl of ~80,000 cpm of either
hIGF-I
dissolved
in
50mM
125
I -WT
sodium
phosphate buffer pH 7.4, containing 0.2 %
BSA, 10 mM EDTA and 0.05 % (w/v)
Tween-20, for 17 h at 4oC. The various
molecular size classes of complexes were
eluted from the column at a rate of 1.2
ml/min using 0.05 M NH4HCO3 buffer pH
7.4. The
125
I content of each 1.2 ml fraction
was counted in a gamma counter.
Tween-20, pH7∙6. The membranes were
blocked in TBS-buffer with Tween with 1%
albumin A2153. The membranes were
incubated
for
one
hour
with
rabbit
antihuman IGFBP-3 1:5000 or polyclonal
rabbit antihuman ALS to the C-terminus.
This was followed with incubation of goat
anti rabbit horseradish peroxidase (HRP)
1:10.000 for one hour. Subsequently, the
membranes were incubated with ECLreagents (Amersham).
Statistics
With exception of IGFBP-1, for the various
parameters
smoothed
references
were
available, based on the LMS method40,
allowing conversion of patients' data
to
SDS values.
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
37
Tabel 1 Plasma levels of the different IGF and IGFBP’s, ALS and Pro-IGF-II. Results from
IGF-I, IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, pro-IGF-II.
Normal range for non-fasting subjects: 24–58 ng IGFBP-1 per milliliter. After an overnight fasting, there is an
average 5-fold rise in normal individuals. After an overnight fasting, normally serum GH is below 20 mIU/L.
serum
IGF-I
IGF-II
IGFBP-1
IGFBP-2
IGFBP-3
IGFBP-4
IGFBP-6
ALS
Pro-IGFII[68-88]
GH
ng/ml
SDS
ng/ml
SDS
ng/ml
Pretreatment During treatment
22 days
83
302
-1,25
4,02
485
743
1,37
4,38
135
<5
During treatment 2:
25 days
225
2,77
701
3,94
26
ng/ml
SDS
mg/L
SDS
ng/ml
SDS
ng/ml
SDS
mU/ml
SDS
ng/ml
1690
4,68
1,38
-0,91
271
1,62
297
1,82
13,45
-0,94
15,2
590
2,33
3,00
2,01
194
0,33
134
-1,54
20,92
1,19
10,4
790
2,96
2,35
1,13
199
0,41
237
0,63
17,75
0,22
13,2
SDS
mIU/L
-0,58
0,91
-2,28
2,5
-1,23
12,0
part of the normal ranges, respectively. The
Results
levels of IGFBP-3 and ALS were between –
Serum levels of ALS, IGFs and IGFBPs
and + 1 SDS (table 1). Serum pro-IGF-
before treatment of NHL and acidosis.
II[68-88] was within the normal range,
strongly suggesting that the tumour cells
Before treatment of NHL and in the
did not produce large amounts of this
presence of acidosis, the concentration of
compound.
IGF-I in patient’s serum was in the lower
range (table 1).
Serum levels of ALS, IGFs and IGFBPs
Circulating levels of IGF-II, IGFBP-4, and
during treatment of NHL and after
IGFBP-6 were in the upper part of the
abolishment of acidosis.
part of the normal
normal range, whereas that of IGFBP-2 was
clearly elevated (table 1). IGF-II in patient’s
The patient was treated successfully with R-
serum was in the lower part and the upper
CHOP
which
also
resulted
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
in
the
38
abolishment of acidosis. Two successive
serum samples were obtained at that time
and the concentrations of GH, ALS, IGFs,
and IGFBPs analysed.
At day 22 during treatment, serum levels of
IGF-I, IGF-II, and IGFBP-3, had increased
markedly
exceeding
the
respective
normative ranges. The concentrations of
serum GH and ALS also increased, albeit to
a lesser extent, and remained within the
normal range. In contrast, serum levels of
IGFBP-1, IGFBP-2, IGFBP-4, and IGFBP-6
had decreased significantly, when compared
to pre-treatment values. The same was
found for pro-IGF-II[68-88] which declined
below – 2 SDS.
At day 25 during treatment, with exception
of the serum levels of GH and IGFBP-4, all
parameters showed changes in the opposite
directions to those observed at day 22.
S200 column chromatography
In patient’s pre-treatment serum 150 kDa
complex formation appeared to be disturbed,
and a higher proportion of [125-I]-IGF-I
was eluted as 40-50 kDa binary complexes
(fig. 10). On day 22, during treatment, the
Fig. 10: A: Distribution of [125]-IGF-I of among the
various molecular size classes in normal human
serum. The complex of 150 kD exists of [125]-IGF-I,
IGFBP-3 and ALS. The complexes of 40-50 kD exist
of IGF-I and various IGFBPs. B: Distribution of
[125]-IGF-I in patient’s serum. Before R-CHOP
treatment, 150 kD complex formation was reduced
when compared both to patient’s serum during
treatment and normal serum.
distribution of [125I]-IGF-I among the
various molecular size classes resembled the
picture seen for normal serum. On day 25
during
treatment,
150
kDa
complex
formation appeared to be slightly decreased
again.
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
39
Analysis of serum IGFBP-3 by Western
bands of lower molecular weight proteolytic
immuno blotting
fragments can be seen. Since the patterns
observed for normal serum and both
The molecular forms of IGFBP-3 in
patient’s pre-treatment and treatment serum
patient’s serum were analysed by Western
were similar it can be concluded that was no
immunoblotting, using a specific antibody
evidence for changes in the proteolysis of
against hIGFBP-3 which recognizes both
serum IGFBP-3.
intact IGFBP-3 and proteolytic fragments.
The results were compared with the pattern
Analysis of serum ALS by Western
immunoblotting
obtained for normal serum. As can been
seen in fig. 11 intact IGFBP-3 migrated as a
doublet with molecular weights of about 40
and 42 kDa, being due to differences in
glycosylation.
Part
of
the
IGFBP-3
migrated with a molecular weight of 30 kDa
due to proteolysis. In addition, some vague
In the control sera two forms of ALS were
clearly identified after Western blotting with
a specific antiserum directed against the Cterminus of hALS, namely at 85 kDa and 90
kDa (data not shown). In patient’s sera, the
same two forms could be identified (fig. 12)
probably represent multimeric (aggregated) forms of
IGFBP-3. Before and after treatment, the distribution
of IGFBP-3 immunoreactivity among intact and
proteolysed IGFBP-3 is similar
Fig. 12 Immunoblot of ALS by using a monoclonal
antibody against the C-terminus of hALS. In all
patients sera two forms of ALS are seen, at 90 and
85 kD ,respectively, representing different
glycosylated forms.
Fig. 11: Immunoblot of IGFBP-3 by using a
polyclonal antibody against hIGFBP-3, recognizing
intact and proteolysed IGFBP-3. Before and after
treatment IGFBP-3 bands of 40 kDa and 42 kDa can
be seen representing different , glycosylated forms of
intact hIGFBP-3. The 30 kDa band represents the
proteolysed fragments. The bands at ~150 kD
Non-Hodgkin lymphoma, its side-effects and the involvement of the IGF-system
Melanie van Berkum, April-July 2011
40
treatment, serum IGFBP-2 levels decreased,
Discussion
but did not normalise.
The subnormal level of IGF-I in patient’s
The levels of ALS in patient’s sera as
serum before treatment could have been be
determined by ELISA were within the
caused by partial GH resistance. Namely, in
normal range. Moreover, no abnormalities
rats it has been found that experimental
of the ALS were observed after Western
acidosis results in GH resistance, leading to
blotting of patient’s sera. Interestingly, 150
reduced hepatic production of IGFBP-3 and
kDa complex formation in pre-treatment
IGF-I15. GH levels of the patient were
serum of the patient was disturbed, as
normal, but it is not known whether the
revealed by S200 column chromatography
expression of the GH receptor by target
after incubation of the serum with 125I-
tissues
IGF-I. The column profile of sera tended to
was
reduced
or
the
signal
transduction pathway disturbed. This can be
normalize during treatment.
evaluated by an IGF-I regeneration test, but
The
this was not done.
complexes could have been caused by the
During treatment, the levels of IGF-I, but
reduction of pH. Acidification leads to
also IGF-II increased dramatically. There is
activation of cathepsin D, a proteinase
not yet an explanation for this finding. It is
which stimulates the proteolysis of IGFBP-
not clear whether the levels in serum will
3, leading to a decreased affinity of IGFBP-
normalise at the long term.
3 to IGF-I, resulting in an increased binding
The level of the precursor of IGF-II, pro-
to IGF-IR. However, this patient did not
IGF-IIE[68-88], appeared to be decreased,
show an increase of proteolysis of IGFBP-3.
and after treatment this level decreased even
Changes in pH will also modify the function
more. Therefore, it is unlikely that pro-IGF-
of ALS. Molecular modelling of ALS at
IIE[68-88] plays a role in the onset of
neutral pH, predicted 20 leucine- rich
disease. Moreover, this patient did not show
repeats arranged in a doughnut-like shape
any signs of hypoglycaemia.
with patches of functional electronegative
The increased level of IGFBP-2 in the pre-
regions within the centre of the cavity
treatment serum of this patient is commonly
where ALS interacts with IGF-IGFBP-3 (or
reduced
formation
of
150
kDa
It has
IGFBP-5) binary complexes. Hence, it may
been proposed that IGFBP-2 may be
well be that at lower pH values the
produced by the malignant cells. During
interaction
seen in patients with leukaemia.
36
of
ALS
with
the
binary
complexes is hampered. As a consequence,
a relatively higher proportion of IGFs is
41
present in binary complexes that may allow
an increased access of IGFs to target cells
including the malignant ones.
This would have an unfavourably effect on
the proliferation of tumour cells. Normally,
insulin
regulates
the
expression
of
hexokinase, a glycolytic enzyme which is
an important enzyme for limiting glycolysis.
IGFs exert insulin-like effects, and also
stimulate the expression of hexokinase,
what results in further deregulation of the
glycolysis and aggravate acidosis5.
Measurements of serum levels of IGFBP-4
and IGFBP-6 by specific RIAs revealed that
during treatment the levels of both proteins
decreased markedly. At present it is not
clear which roles these IGFBPs play in this
disease picture. Acidosis increased the
expression of IGFBP-4 in cultured murine
mandibular
condyles41.
In
addition
increased levels of IGFBP-4 have been
found in patients with acute lymphoblastic
leukaemia28. Further investigation is needed
to understand the involvement of the IGFsystem
in
complicated
non-Hodgkin
by
acidosis.
lymphoma
This
may
contribute to an effective treatment of this
disease.
42
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