Download Long term in vitro-cultured plant cells show typical neoplastic

Biology of the Cell 95 (2003) 357–364
www.elsevier.com/locate/bicell
Long term in vitro-cultured plant cells show typical neoplastic features at
the cytological level
Julien Häsler a,*, Jean Wüest b, Thomas Gaspar a,c, Michèle Crèvecoeur a
a
Laboratoire de biochimie et physiologie végétales, université de Genève, place de l’Université 3, 1211 Genève 4, Suisse
b
Muséum d’histoire naturelle, route de Malagnou 1, 1208 Genève, Suisse
c
Biologie moléculaire et hormonologie végétales, université de Liège, Sart Tilman B-22, 4000 Liège, Belgique
Received 28 April 2003; accepted 23 June 2003
Abstract
Cells from a green normal (dependent on exogenous hormones) callus and from an achlorophyllous fully habituated (independent from
exogenous hormones) callus, both generated from the same sugarbeet strain more than twenty years ago, were reexamined cytologically, ten
years after the first comparative description. Cells from the habituated callus, already considered as neoplastic cells, because terminating a
neoplastic progression where the organogenic totipotency was lost, still showed nuclear invaginations, polynucleolation, vacuolation of
nucleoli and incomplete cell walls, nevertheless at a higher degree. The present study particularly shows that, compared to their previous
description, normal cells have started to acquire some features (polynucleolation, nuclear invaginations...) that are typical of the neoplastic
cells. This suggests that normal cells, on the long term, also entered a neoplastic progression, which should explain the known progressive loss
of regeneration capacity of too long subcultured hormone-dependent calli.
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Habituation; Plant cancer; Nucleolar vacuole; Incomplete cell wall; Callus
1. Introduction
Tumorigenesis has been described in animal cells as a
multistep process. These steps modelize genetic alterations
driving the progressive transformation of normal cells into
highly malignant derivatives. The process involved is analogous to Darwinian evolution in which a succession of genetic
changes, each conferring a growth advantage or another,
leads to the progressive conversion of normal cells into
invasive cancer cells. The first phase of this transformation
involves the alteration of an oncogene, under the effect of an
endogenous or exogenous carcinogenic agent. This results in
the development of a primary benignant tumor which grows,
while DNA mutations still occur, and becomes malignant as
soon as it releases invasive circulating metastases. This progression from normal to cancerous state is accompanied by
an array of morphological and physiological changes at the
cellular level such as self-sufficiency for growth signals,
* Corresponding author. Département de Biologie Cellulaire Sciences
III 32, Boulevard d’Ivoy 1211 GENEVE-4 Suisse FAX: (+41 22) 379 64
42
E-mail address: [email protected] (J. Häsler).
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
doi:10.1016/S0248-4900(03)00077-7
reduction of cell-to-cell adhesion, polyploidy, aneuploidy
and high rate of cell division (for a review see (Hanahan and
Weinberg, 2000)). This anarchic cell proliferation also takes
advantage of a parallel loss of cell differentiation and of
localized specific function in a tissue. The whole process of
transformation of normal cells into cancerous daughter cells
is often termed neoplastic progression.
Tumorous growth has been described for plant cells (usually induced by pathogens) with many morphological and
biochemical features similar to those found in animal cells
(Braun, 1978). However the concept of plant cancer and of
plant cancerous cells remains rather vague. It was even
claimed that plants could not get cancer, the main reason
being the absence of circulating metastases (Doonan and
Hunt, 1996). We are convinced that the problem is a simple
question of concept and of adapted definition (Gaspar, 1998).
The process of habituation is defined as a stable heritable
loss of requirement of cultured plant cells for growth factors.
Habituated sugar beet calli have been obtained by hormonesand cold-treatments (De Greef and Jacobs, 1979). Two of
these cell lines have been subcultured so far in the same
conditions and extensively studied (for a review see (Gaspar
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Table 1
Characteristics, which indicate that cells from HNO callus are cancerous
cells
MORPHOLOGY
Deficiency in cell wall differenciation
Deficiency in chloroplast and mitochondria
differenciation
Nuclei with irregular shape and many
nucleoli
BIOCHEMISTRY
Hyperhydricity
Deficiency of tetrapyrrole-containing
compounds
Permanent oxydative stress
Accumulation of polyamines
CELL PHYSIOLOGY
Independence to growth regulators
Polyploidy and aneuploidy
Reduced cell-to-cell adhesion
Susceptibility to necrosis
TYPICAL PLANT CANCER TRAIT
Loss of organogenic totipotency
(Crèvecoeur et al., 1992)
(Crèvecoeur et al., 1992)
(Hagège et al., 1992a)
(Crèvecoeur et al., 1987)
(Hagège et al., 1992b)
(Arbillot et al., 1991)
(Hagège et al., 1990)
(De Greef and Jacobs, 1979)
(Kevers et al., 1999)
(Liners et al., 1994)
(Kevers et al., 1995)
(Gaspar et al., 2000)
et al., 2000)). The first cell line is a green normal callus called
N, which is still fully dependent of hormones (auxin and
cytokinin) for its growth; the second one is a white habituated
callus of the same species/strain, which is fully independent
of hormones for its growth and has completely lost its organogenic capacities. It is so designated HNO for Habituated
Non-Organogenic.
Numerous previous studies have compared these two cell
lines at the biochemical, morphological and physiological
levels. The HNO cells show numerous features, summarized
in Table 1, which are those of animal metastases. They
include close to full hormone independence, polyploidy and
aneuploidy, loss of cell-to-cell adhesion essentially due to an
over esterification of pectins, a permanent oxidative stress
and accumulation of polyamines.
These characteristics have been used to develop the concept of “in vitro plant cancer” which has been previously
reviewed (Gaspar, 1999). HNO cells represent the ultimate
step of a neoplastic progression from normal N cells to
highly cancerous HNO cells. Plant cancer cells evidently
cannot become invasive metastases, but because normal
plant cells, unlike animal cells, have the unique capacity to
organize themselves into organogenic or regenerating meristems, the typical plant cancer trait has been precisely defined as the irreversible loss of organogenic totipotency i.e.,
the incapacity for such cells to reorganize primary organogenic meristems at the end of a neoplastic progression. This
definition makes a clear distinction from plant tumors (such
as those mediated by pathogens or resulting from genetic
transformation) which are chimaeric and still organogenic
(Gaspar, 1999).
In this study, we give another morphological and cytological description of N and HNO cell lines. These cells have
been maintained in in vitro culture for twenty-four years and
the last histological studies describing them have been performed around 10 years ago (Crèvecoeur et al., 1992).
Surprisingly, we observe that, after more than twenty
years of in vitro culture, the N cells, which still necessitate
growth regulators for their proliferation, start to show, at the
cytological level, some features that were typical of the HNO
cancerous cells. The present work then suggests that, after
long term in vitro culture, N cells are spontaneously entering
into a neoplastic progression leading to cancerous state.
2. Results
The morphological aspect of sugar beet calli at the 11th
day of culture on solid agar medium is shown in Fig. 1. The
most pronounced visual difference concerns the color i.e.
green for normal callus and white for the habituated callus.
Scanning electron microscopy revealed that cells from both
calli were heterogeneous in size and appearance. Their length
varies from 100 to 200 µm. In the N callus, most of the cells are
spheroid but some of them present an elongated shape
(Fig. 2A). HNO cells are most generally spheroid (Fig. 2B).
No elongated cells have been observed in HNO calli. Observations of samples at higher magnification revealed the presence of two types of protuberances at the surface of cells from
both calli. The first type of protuberance is large and spherical
Fig. 1. N (A) and HNO (B) calli growing on their solid medium eleven days after subculture.
J. Häsler et al. / Biology of the Cell 95 (2003) 357–364
Fig. 2. Scanning electron micrographs of N and HNO cells.
A Overview of a N callus. Numerous cells show an elongated shape.
B Overview of a HNO callus. Cells are mostly spheroid.
C-D Details showing “buds” on the surface of HNO cells.
E “Hernia” on the surface of a N cell.
F “Hernia” on the surface of a HNO cell.
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and is called “budding” (Fig. 2 C-D). The second one is
smaller and strangulated and is called “hernia” (Fig. 2 E-F).
Examination of Historesin sections showed that the nuclei
react very poorly with toluidine blue in N cells whereas they
were densely stained in HNO ones (Fig. 3 A-B). The shape of
nuclei in N cells is generally round, with small invaginations
in 25% of the cells. The shape of the nuclei is much more
irregular in HNO cells. They present more frequent and
deeper invaginations (Fig. 3D) in 70% of the cells. However,
the measurement of the nuclear surfaces did not indicate a
significant difference between normal and habituated calli. In
both cell lines, approximately 60% of the nuclei have a
nuclear surface varying from 130 to 375 µm2. The distribution of nuclear surface values for the two cell lines appeared
very similar, with perhaps a slight tendency to a higher
proportion of smaller values in HNO cells and of larger in the
N ones (Fig. 4).
Another nuclear difference between the two cell lines
concerns the number and the structure of the nucleolus. A
morphometric analysis has been performed to estimate these
observations. In the N callus only one nucleolus per nucleus
was found in 90% of cells while 80 % of nuclei in HNO calli
contained more than two nucleoli (Table 2). The number of
nucleoli in the HNO nuclei is highly variable, with a number
as important as 18 in a few cells. Nucleolar vacuoles, that
were sometimes particularly large (Fig. 3C), have been observed in both calli. One of the most noticeable histological
differences between the two cell lines was the presence of
xylem cells in N calli that were clearly stained in turquoise
with toluidine blue (unshown data). They have never been
observed in sections from HNO calli. Examination of serial
sections showed that elongated cells of the N callus are
composed of two distinct cells, each containing a nucleus
(Fig. 3E). Incomplete cell walls growing into the cytoplasm
were observed in N and HNO cells (Fig. 3 F-G).
Staining of living cells with lugol, a reactive used for starch
localization, is shown in Fig. 5 A-D. HNO cells cultured in
liquid medium were strongly stained in brown by lugol. Numerous dense dark aggregates were observed in the cytoplasm
(Fig. 5B). Observations at higher magnification indicated that
they are contained in large granules (Fig. 5D). N cells were
poorly stained by lugol (Fig. 5A) and an observation at higher
magnification indicated that starch is accumulated in smaller
and less numerous granules in this cell type (Fig. 5C).
Transmission electron microscopy revealed several cytological features in greater details. The irregular shaped nuclei
with several nucleoli in HNO cells is illustrated in Fig. 5E. In
the cytoplasm of these cells a great number of plastids containing several very large starch grains and very few thylakoids were observed (Fig. 5F).
3. Discussion
As compared to the last histological descriptions that have
been made ten years ago, the N and HNO cells present
several new characteristics.
First of all, the two types of protrusions present at the
surface of the cells (buds and hernia) have been described
earlier as present only on HNO cells (Hagège et al., 1991).
The present work has shown that today, these two types of
protuberance are present on the surface of both cell lines.
Their function is still unknown but some unshown pictures
strongly suggest that the buds sometimes contain an accessory nucleus. These protuberances could then be the result of
an atypical incomplete cell division. Mitotic figures are indeed very rare and this could suggest that the biomass increase of the calli is more based on this type of atypical,
partial or complete, cell divisions than on regular mitosis.
Nuclei react very poorly with toluidine blue in N cells
whereas they were densely stained in HNO ones. Toluidine
blue is a well known cationic dye that is attracted by cellular
components that are acidic in nature. Nucleoli that are rich in
nucleic acids appear generally densely blue stained with this
stain. Nuclei in habituated calli appear more densely stained
probably because of the higher number of nucleoli per
nucleus. The nuclear invaginations, the polynucleolation and
the vacuolation of nucleoli are representative features of a
very high rate of cellular metabolism, often observed in
cancerous cells. The function of nucleolar vacuoles remains
unclear but they have been observed in a large field of animal
and plant tissues in different physiological states (Chouinard,
1982; Feldman and Torrey, 1977; Lai and Srivastava, 1976;
Tumanishvili and Chelidze, 1983) and in maize calli (Fransz
and Schel, 1987). Their function has been interpreted as a
transient storage area for ribosomal ribonucleoprotein that
could facilitate their transport by increasing the
nucleus/nucleolus interface (Jennane et al., 2000; Medina et
al., 2000). The invaginations of the nucleus could assume an
analogous function of transport in highly metabolic cells by
increasing the nucleus/cytoplasm interface. These features
have been described earlier as specific of the HNO cell line
(Crèvecoeur et al., 1992), but in the present work they have
been observed on both cell types even if they are more
frequent and numerous on HNO cells. Also, the nuclear
surfaces previously described as much higher in HNO cells
than in N ones (Hagège et al., 1992) seem to be, today, quite
similar in both cell lines. The number of nucleoli is still much
higher in HNO but a certain number of N cells start showing
this feature now.
The presence of incomplete cell walls growing into the
cytoplasm has been described earlier as specific of the HNO
cells too (Crèvecoeur et al., 1992), but the present study
shows that these ingrowths are now present in N cells as well
as in HNO cells. The role of such cell wall ingrowths in the
cytoplasm is unclear but they have already been observed in
cells, which are deficient in cellulose synthesis (Keller et al.,
1994; Sabba et al., 1999; Sabba and Vaughn, 1999).This
correlates well with earlier studies showing that the HNO cell
walls contains very few lignin and cellulose (Crèvecoeur et
al., 1987).
Finally, the starch detection by lugol staining and transmission electron microscopy shows that the HNO cells con-
J. Häsler et al. / Biology of the Cell 95 (2003) 357–364
361
Fig. 3. Toluidine blue staining of historesin sections of N and HNO cells.
A Section of N callus stained with toluidine blue. The nuclei reacted very poorly with toluidine blue and their shape is generally round.
B Section of HNO callus stained with toluidine blue. The nuclei are strongly stained by toluidine blue; most of them are lobed with deep invagination and contain
many nucleoli.
C HNO cell containing a highly Vacuolated Nucleolus (VN). Note the presence of numerous vacuoles in the cytoplasm (V).
D HNO cells showing highly invaginated nuclei containing numerous nucleoli.
E Section of an “elongated” N cell. The “cell” is composed of two distinct cells with independent nuclei (N) separated by a cell wall (CW).
F Incomplete cell wall (icw) growing into the cytoplasm of a N cell.
G Incomplete cell wall (icw) growing into the cytoplasm of a HNO cell.
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that cancerization still is much more complex than being a
simple ontogenic retrogradation or rejuvenation process.
4. Materials and methods
4.1. Tissue and cell culture
Fig. 4. Distribution of nuclear surfaces in N and HNO cells.
250 cells have been randomly chosen in each cell line on historesin section
stained with toluidine blue. Nuclear surfaces have been measured using a
quantimet 500+ (Leica).
tain more starch in their plastids, which is stored in much
bigger granules, than the N cells. This result is in contradiction with earlier studies showing that the N plastids contain
more starch than the HNO ones (Crèvecoeur et al., 1992).
Taken all together, these data strongly suggest that dramatic metabolic changes occurred in N and HNO cells over
the last ten years. The N cells start showing some features
like polynucleolation or invagination of the nuclei that were
typical of the HNO cancerous cells. This suggests that after
more than twenty years of in vitro culture, N cells are
spontaneously entering in a neoplastic progression leading to
the HNO state. Despite many attempts, they have never been
able to grow in the absence of growth regulators. However,
they became progressively unable to regenerate any organ
type under a modified auxin/cytokinin balance, which they
were able to do in the primary callus and during the first
subcultures. This progressive loss of regeneration capacity
on the long term was known but without definitive explanation (Gaspar et al., 2000).
The next part of this work will obviously be to update the
biochemical data concerning both cell lines to confirm that
the N cells become cancerous.
Even if the cytological differences observed when compared to the last histological studies are more pronounced for
the N cells, the HNO ones seem to advance in the neoplastic
progression too. Starch detection indeed reflects that starch
metabolism changed in both cell types and cancer features,
like invagination of the nuclei and polynucleolation, seem to
be now more pronounced in HNO cells.
As organisms are more exposed to cancer as they age, due
to spontaneous mutations and the endogenous carcinogenic
effect of the environment, it is not very surprising to see cells
that have been cultured in vitro for more than twenty years
becoming cancerous. In any case, the present results indicate
The hormone requiring (N) and hormone independent
(HNO) calli have been obtained from sugar beet leaves explants (Beta vulgaris L. ssp altissima cv F3S52, diploid
strain from the Belgian factory SES, Tienen) as previously
described (De Greef, 1978). They have been maintained by
successive subcultures on their respective solid media: basal
medium without growth regulators in the case of HNO calli
and basal medium supplemented with 0.1mg.L-1 2,4-D and
0.1 mg. L-1 BAP in the case of N calli. Both calli are grown at
25°C under light (16 h photoperiod of Sylvania Grolux fluorescent light providing 17 Wm-2).
Suspension cultures of N and HNO lines are grown in
250 ml Erlenmeyer flasks in the same medium as calli, but
without agar. They are grown under rotary shaking (130 rpm)
in the same light and temperature conditions as calli. Calli
and cell suspensions are subcultured every 14 days. All the
histological and cytological observations have been performed at the 11th day of culture.
4.2. Tissue processing for light microscopy
Samples were fixed 12 hours at 4°C by immersion in a
mixture of 3% paraformaldehyde and 0.3% glutaraldehyde
in 20 mM phosphate-buffered saline (PBS) (pH 7.2). Fixed
samples were rinsed several times in PBS and dehydrated
through a graded ethanol series from 25% to absolute ethanol. They were finally embedded into Historesin (Reichert
Jung). Sections, 5 µm thick, were made with a glass knife on
a rotative microtome, and picked up on slides. They were
stained by immersion in toluidine blue (0.1% in 2.5%
Na2CO3) at 55°C for 20 minutes.
Starch detection has been performed in non-fixed cells by
direct staining in a 1% KI - 0.5% I2 solution for 1 minute at
room temperature.
Cells and sections were observed with a Zeiss Laborlux
light microscope and photographed with a Leica numeric
camera DC 100.
Nuclear morphometric analysis has been performed on
250 randomly selected cells of each cell line with a Quantimet 500 + (Leica).
Table 2
Distribution of the number of nucleoli per nucleus in N and HNO cells.
Nucleoli have been counted in 250 individual nuclei of each cell type, randomly chosen on historesin sections stained with toluidine blue.
Number of nucleoli per nucleus
1
N cells
88%
HNO cells
12%
2
9%
14%
3
2%
20%
4
1%
15%
5
0%
11%
6
0%
11%
>6
0%
17%
J. Häsler et al. / Biology of the Cell 95 (2003) 357–364
Fig. 5. Starch detection in N and HNO cells by lugol staining and transmission electron microscopy.
A Living N cells (from cell suspension) stained with lugol. Cells are poorly colored.
B As A with HNO cells. Cells show a strong black staining.
C Detail of N cells stained with lugol. Starch is accumulated as small aggregates.
D Detail of HNO cells stained with lugol. Starch is accumulated as big aggregates.
E Transmission electron micrograph of a HNO cell showing a highly invaginated nucleus and many amyloplasts containing huge starch grains (A).
F Detail of E showing starch grains in amyloplasts.
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4.3. Tissue processing for scanning electron microscopy
Samples were fixed for 16 hours in 4% glutaraldehyde
prepared in 100 mM phosphate buffer (pH 7.2). After several
rinses in phosphate buffer, the samples were dehydrated in
ethanol, infiltrated in amyl acetate before immersion in liquid
CO2. They were critical point dried and sputter-coated with
gold. Scanning electron microscopy was performed using a
Zeiss DSM 940A electron microscope.
4.4. Tissue processing for transmission electron
microscopy
Samples were fixed for 3 hours at 4°C in 4% glutaraldehyde buffered to pH 7.2 with 100 mM phosphate buffer. They
were washed and postfixed for 2 hours at 4°C in 1% osmium
tetroxide prepared in the same buffer. After several rinses, the
samples were dehydrated in ethanol and embedded in Epon.
Ultrathin sections (90 nm thick) were stained classically with
2% uranyl acetate in water for 30 minutes followed by lead
citrate (Reynolds, 1963) for 15 minutes and examined with a
Phillips EM 410 electron microscope operating at 60 kV.
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