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RAPID COMMUNICATION
Homozygous Loss of the Cyclin-Dependent Kinase 4-Inhibitor (p16) Gene
in Human Leukemias
By Seishi Ogawa, Naoto Hirano, Naomi Sato, Tokiharu Takahashi, Akira Hangaishi, Kozo Tanaka, Mineo Kurokawa,
Tomoyuki Tanaka, Kinuko Mitani, Yoshio Yazaki, and Hisamaru Hirai
Recently, it has been shownthat the homozygous deletion
of the cyclin-dependent kinase-4inhibitor (CDK41; p161 gene,
which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4 gene is thoughtto be a putative tumor-suppressor gene. We report here
that both alleles
of the CDKU gene were completely or partially deleted in
human leukemia cells derived
from both patients and established cell lines. Thirty-seven hematopoietic cell lines and
samples from 72 patients with leukemias were examined
for homozygous loss of the CDK4l gene locus by Southern
blot analysis. We found that a part or the whole of the CDK41
gene was homozygously deleted in 14 of the 37 (38%) cell
lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute
lymphocytic leukemia(ALL), and 13with chronic myelocytic
leukemia in blastic crisis. In the cell lines, the homozygous
deletion of the CDK4l gene was detected in a variety of cell
lineages, whereas all
4 cases showingthe homozygous deletion were confined to ALL. It should be noted that 2 of them
of chromosome 9. Our rehad no cytogenetic abnormalities
sults suggestthat loss of the CDKU function may contribute
to immortalizationof human leukemiac e l l s and play a causative role at least in development of human lymphocytic
leukemias.
0 1994 by The American Societyof Hematology.
10% fetal calf serum. TFl, W, CMK, and F36E are factor-depenANY LINES OF evidences have established the sigdent cell lines and are cultured in the presence of 5 ng/mL of recomnificance of inactivation of tumor-suppressor gene(s)
binant human granulocyte-monocyte colony-stimulating factor
in development of human malignancies, including leuke(rhGM-CSF).
mias.I4 Many investigators have reported mutations andor
Patients and preparation of sample cells. Bone marrow or pedeletion of p53, RB, WT1, and NF1 genes in a wide variety
ripheral blood samples from 72 patients, including 45 patients with
of both familial and sporadic cases of human cancer~.~*~-’lacute myelocytic leukemia (AML), 14 patients with acute lymphoThese genetic abnormalities are considered to deteriorate
cytic leukemia (ALL), and 13 patients with chronic myelogenous
normal functions of the tumor-suppressor gene products
leukemia (CML) in blastic crisis, were collected after informed conquantitatively or qualitatively and to make a contribution to
sent was obtained. In all samples examined, the proportion of tumor
carcinogenesis.’” The cell cycle in eukaryotes is regulated
cells exceeded 70%. From the samples, mononuclear cells were
separated on Ficoll-Hypaque density gradients.
by the cyclin-dependent kinases (CDKS).”*’~ The
sequential
Synthetic primers. The reverse transcription polymerase chain
activation of CDKs and their cosequent phosphorylation of
reaction (RT-PCR) primers were designed to amplify almost the
critical substrates stimulates progressionofthe
cell
entire coding sequence (nucleotide [nt] 32 to 485) ofthe CDK4I
The complexes formed by CDK4 and the D-type cyclins
cDNA previously reported by Serrano et ai?’ Their sequences are
control passage through the G1 phase of the cell
as follows: the antisense primer for RT (nt 526 to 507), 5‘-AGGThe CDK4-inhibitor (CDK4I; p16) is a protein of 16 kD
ACCITCGGTGACTGAT-3’; the primers for the first PCR, 5’that binds to and inhibits the catalytic activity of the CDK4/
CAGCATGGAGCCITCGG-3’ (sense) (nt 15 tont 31) and 5’-TCTcyclin D c ~ m p l e x e s . ~Because
’ - ~ ~ CDK4I is a negative reguAAGTITCCCGA-GGTITC-3’ (antisense) (nt 505 to nt 486); and
lator of cell-cycle progression, it has been thought to be
the primers for the second PCR, 5”CTGACTGGCTGGCCAinactivated during cancer d e v e l ~ p m e n t Recently,
.~~
Nobori
CGGCC-3’ (sense) (nt 32 to nt 51) and5’-TCAGAGCCTCTCTGGTT%TT-3’ (antisense) (nt 485 to nt 466).
et alZ4and Kamb et al” have proposed that the CDK4I gene
RT-PCR analysis. Total RNA was isolated from normal bone
is a novel candidate of a tumor-suppressor gene. They have
marrow mononuclear cells with the single step method described by
reported that the CDK4I gene is deleted or mutated with
Chomczynski and Sacchi.26 Five hundred nanograms of total RNA
surprisingly high frequencies in human cancer cell lines,
was mixed in a reaction mixture containing 2 pmol of RT primers
including melanoma, glioma, lung cancers, and leukemias.
for the CDK4I gene, 3.8 pL of 5X RT buffer (250 mmoVL TrisThese findings prompted us to investigate the existence of
HCI [pH 8.31, 375 mmol/L KCI, 15 mmoVL MgCI,) in a volume of
homozygous loss of this gene in samples from patients with
12.5 pL. The mixture was heated at 95°C for 3 minutes, chilled on
leukemias as well as in human hematopoietic cell lines by
Southern blot analysis.
M
MATERIALSANDMETHODS
Cell lines. Thirty-seven human leukemia cell lines were included
in this study and consist of 11 myelocytidmonocytic cell lines (KG1, HL60, HEF-2, KU8 12, SKHI, JOSK-I, JOSK-K, JOSK-S, P31
FUJ, P39 TSU, and THP-l), 4 erythroid cell lines (TF-I, F36E,
HEL, and K562). 4 megakaryocytic cell lines (W,CMK, MegJ,
and MOLMI), 1 1 B-lymphocytic cell lines (SCMCL-Ll, SCMCLL3, SCMCL-LA, BALLl, P32 ISH, P30 OHK, I“9, Daudi, HA,
M5, and Raji), and 7 T-lymphocytic cell lines (MOLT16, Jurkat,
MT2, CEM, MOLT4, SKW3, and A3KWA). The cells were grown
in suspension culture inRPM1 medium 1640 supplemented with
Blood, Vol 84, No 8 (October 15), 1994 pp 2431-2435
FromThe Third Department of Inteml Medicine, Faculty of
Medicine, University of Tokyo, Tokyo, Japan.
Submitted June 23, 1994; accepted July 22, 1994.
Address reprint requests to Hisamaru Hirai, MD, The Third Department of Inteml Medicine, Faculty of Medicine, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advettisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1994 by The American Society of Hematology.
0006-4971/94/8408-06$3.00/0
2431
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OGAWA ET AL
2432
with Hindnl, separated through 0.8% agarose gels, and blottedonto
the nylon filters. The RT-PCR products of CDK41 cDNA were excised from low melting temperature agarose gels, extracted from the
gels with phenolkhloroform. and used as a probe for detecting the
a kindgift
CDK4IDNA. A humanAMLlprobe,C6E3SS2,was
A
kb
23
9.4
6.6
-
Table 1. Homozygous Loss or Rearrangement of the CDK4l Gene
in Hematodetic Cell tines
B
kb
23
9.4
6.6
1
-
"
"
"
-
2
3
4
5
6
7
8
9 1 0 1 1 1 2
~
- ""~r"""
I
*
Fig 1. Southern blot analysis of human hematopoietic c
e
l lines.
The CDK4 gene was detected as the threediscrete bands of approximately 20, 12, and 6 kb in size by Hindlll digestion of genomic DNA.
The AMLl gene fragment of 7 kb in size was hybridized as an internal
control and indicated by an arrow. (A) Lane 1, Jurkat; lane 2, MOLT4;
lane 3, SCML-L4; lane 4,MOLT%; lane 5, SCML-U; lane 6, SKW3;
lane 7, JOSK-S; lane 8, MT2; lane 9, P39-TSU; lane 10, P3O-OHK; lane
11, TF1; lane 12, normal bone marrow.(B) Lane 1, KG1; lane 2, MP1;
lane 3, SKH1; lane 4,HEL; lane 5, CMK; lane 6, MegJ; lane 7, KU812;
lane 8, UT7; lane 9, F36E; lane 10, K562; lane 11, M5; lane 12, JOSK1.
Myelocytidmonocytic cell lines
HL60
SKHl
KG1
KU812
HEF2
JOSK-I
JOSK-K
JOSK-S
P39 TSU
P31 FUJ
THPl
Erythroid cell lines
F36E
K562
HEL
TF1
Megakaryocytic cell lines
UT7
CMK
MegJ
MOLMl
B-lymphocytic cell line
BALL1
Daudi
Raji
IM9
M5
HA
P32 ISH
SCMCL-L1
SCMCL-L3
SCMCL-L4
P30 OHK
ice, and incubated at 37°Cfor 15 minutes for annealing. The mixture
was then supplemented with 0.2 pL of 5 X RT buffer, 2 pL of 0.1
moVL D'IT, 4.0 pL of 2.5 mmol/L each of dNTPs, 20 U of RNase
inhibitor(Takara,Kyoto, Japan), and 1 0 0 U ofMoloneymurine
BRL, Gaithleukemia virus (M-MLV) reverse transcriptase (GIBCO
ersburg, MD) to a volume of 20 pL and incubated at 37°C for 60
minutes. The RT reaction products were again heated at 96°C for 2
minutes and chilled on ice. The 2 pL of the RT reaction products
was subjected to the first PCR amplification in
50 pL of reaction
mixture containing IO mmol/L Tris-HCI (pH 8.8). 50 mmoVL KCI,
3.5 mmol/L MgC12,0.01% bovine serum albumin (BSA), 200 pmoV
L dNTPs, 5% dimethylsulfoxide, 15% glycerol, I pmoVLeachof
T-lymphocytic cell lines
thefirst PCRprimers,and2.5
U TaqDNA polymeme (PerkinJurkat
Elmer Cetus, Norwalk, CT). The PCR were repeated for 40 cycles
of96°C (1 minute), 50°C (1 minute),and72°C (2 minutes). Two
A W W
MOLT4
microliters of the first reaction products was subjected to the nested
MOLT16
second PCR reaction, essentially under the same conditions as the
SKW3
first reaction except that the
pH of the buffer was 9.3, the concentraCEM
tion of MgC12 was 1.5 mmol/L, and the primer set for the second
MT2
PCR was used.
Sequencing of RT-PCR products. For subsequent use as a probe
for Southern blot analysis, the RT-PCR product of the CDK4l cDNA
Total
from the normal bone marrowsample was subcloned into pBluscript
Abbreviations: DD, neither of both alleles of the CDK41 gene were
SK(-) (Stratagene, La Jolla, CA) andsequencedby the dideoxy
detected; Dm, solely a rearranged allele was detected, indicating loss
chain termination method.
of the CDK41 genein one allele and a rearrangement in another allele;
Southern hlor analysis. Ten micrograms of DNAs extracted from
cultured leukemia cell lines and from patients' samples was digested -, negative for homozygous loss of the CDK41 locus.
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CDK41 ( ~ 1 6 GENE
)
IN HUMAN LEUKEMIAS
2433
1
2
3
4
"
"
5
6
7
8
9 1 0 1 1 1 2
-
kb
23
9.4
6.6
4.4
2.3
2.0
"
9
--
"
"
"
"
"
"
I
C
4-
Fig 2. Southem blot analysis of samples from patients with leukemias. Lanes l and 2, normal bone
marrow; lanes 3 through 6, patients with ALL showing homozygous loss or rearrangement of the
CDK491 gene; lanes 7 through 9, patients with AML;
and lanes l 0 through 12, patients with CML in blastic
crisis. The AMLl gene as an internal control was indicated by an arrow.
from Dr H. Miyoshi (Radiobiology Division, National Cancer ResearchInstitute.Tukiji.Tokyo,Japan)and
was usedtoconfirm
integrity of the sample DNA and completeness of Hind111 digestion.
The 25 pg of both probes was labeled to high specific activities
with [(r--'*P]dCTF'by the random priming method and used as probes
for detection of the Hindlll-digested fragments of the CDMI and
AMLlgenomic DNA. Hybridimtionwasperformed in a solution
with 50% formamide, 5 X SSC, 5 X Denhart's solution, and 0.5%
sodium dodecyl sulfate (SDS)at 42°C overnight. After highly stringent wash with 0.1X SSC and 0.1% SDS at 65°C. filters were exposed to x-ray films with an intensifying screen at -70°C.
RESULTS
Sequencing of the RT-PCR product. A 463-bp cDNA
fragment was amplified using RNA from normalhuman
bone marrow cells with the RT-FCR method and confirmed
to be the authentic CDK4I cDNA fragment by nucleotide
sequencing?'
Southern blot analysis of DNAs of hematopoietic cell lines
and samples from leukemia patients. We first analyzed 37
leukemia cell lines for the configuration of the CDK4I gene
locus (Fig 1A and B). The normal CDK4I genomic DNA
Table 2. Homozygous Loss of the CDK41 Gene
in Blood Samples of Leukemia Patients
Homozygous Loss of
the CDKIl Gene
Disease
0145
AML
ALL
CML-BC
Total
4/14
0113
(5.6%)
Abbreviation: CML-BC, CML in blastic crisis.
4/72
was detected as three distinct bands of approximately 20,
12, and 6 kb in size, whereas in 14 of 37 cell lines (38%).
one or more of the three bands for the CDK4I gene failed
to be detected. Because the AMLl probe could successfully
detect a 7.0-kb HindIII genomic fragment in every lane, it
was confirmed that all sample DNAs examined were highly
intact and completely digested with HindIII. Therefore, it is
concluded that all or a part of the CDK4I gene locus was
homozygously deleted in these cell lines. In lanes 1, 4, and
IO ofFig 1A and in lanes 2, 4, 6, 8, 9, and 10 of Fig
IB, we could not detect any of the three HindIII-digested
fragments of the CDK4I gene with this probe (a 17-kb band
seen in most lanes of Fig 1A is a nonspecific band), whereas
in lanes 2 and 6 of Fig I A, the partially deleted CDK4 gene
fragments with or without rearrangement were observed.
These observations were confirmed by digestion with another restriction enzyme, EcoRI, suggesting that the deleted
region of the CDK4I gene locus is variable among cell lines.
The results were summarized in Table 1, which suggests
that, in the cell lines analyzed, the homozygous deletion of
the CDK4I gene was detected in a variety of cell lineages.
To test the possibility that the homozygous deletion could
also occur in leukemia cells from patients, we examined the
blood samples from 72 leukemia patients. Because, in contrast to cell lines, patients' blood samples inevitably contain
residual normal bloodcells and thus introduced normal DNA
into the prepared sample DNA,whichmight
disturb the
accurate evaluation of the results obtained by Southern blot
analysis, we have included only the samples that were assessed to contain more than 70% leukemic cells so that the
DNA of normal cells could be neglected or properly evaluated by using the internal control, ie, the AMLl gene. Taking
this into consideration, it was concluded that, in 4 of 14
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2434
OGAWA ET AL
Table 3. Clinical and Cytogenetic Characteristics of 4 Patients With Loss of the CDK41 Gene
Patient No.
Age (yr)/Sex
Diagnosis
Blast (%l
1
2
3
4
2/F
6OlF
30lM
52/F
Null ALL
Pre-B ALL
Null ALL
Pre-B ALL
90.0
88.8
93.6
98.6
Karyotype
+8,51,
+7,XX,
del(l)(q32),
f21,
46, XX, -1. -4, 2q-, 5q+, 6p-,
41, XY,
-6,
-6, -9,
-13,
-17,
-22,
46,XX. -18, l q - , 13q-,
4p+,
samples from ALL patients, homozygous loss of a part or
the whole of the CDK4I gene was detected, compared with
the AMLl band being clearly detected (Fig 2). However, in
the other samples from patients, including 45 cases with
AML and 13 cases with CML in blastic crisis, we could not
detect the homozygous deletion or rearrangements of the
gene (Table 2). As for the cell lineages of the 4 ALL cases
with the homozygous deletion of the CDK4I gene, 2 are preB ALL and the other 2 are null ALL. Despite the CDK4I
gene locus on the short arm of human chromosome 9, 2 of
the 4 cases did not show any cytogenetical abnormalities
involving chromosome 9.
DISCUSSION
The cell division cycle is regulated by a number of protein
kinases known as CDK4s,””‘ among which the CDK4 is
considered to associate with the D-type cyclins and to control
cell proliferation through the G1 phase.”-” The CDK4I was
originally identified as a protein of relative molecular mass
of 16 kD (p16), which associates with CDK4 and inhibits
catalytic activity of the CDK4kyclin D complexes and cell
proliferati~n?~.~~
In this context, the recent reports that the
CDK4I gene is homozygously lost in a wide variety of human tumor cell linesz3-z5seem to strongly support the idea
that it is a tumor-suppressor gene that had long been quested
for in the 9p21 locus, a frequently deleted chromosomal
region in ALL and other various t ~ m o r s . ~For
~ ” ~further
confirmation of the idea, therefore, we analyzed 72 leukemia
patients as well as 37 leukemia cell lines for loss of the
CDK4I gene by Southern blot analysis. As a result, it is
shown that at least a part of the CDK4I gene was homozygously lost, witha surprisingly high frequency (38%) in
hematopoietic cell lines. Considering that the incidence of
mutations of both p53 alleles is estimated around 75% in
human leukemia cell lines (Cheng and H a a ~ Sugimoto
,~~
et
al,” and our unpublished observations), this suggests that
the inactivation of the CDK4I gene may similarly be important for cell immortalization as that of the p53 gene.
Moreover, inactivation of both CDK4I and p53 occurred in
THP1, F36E, K562, UT7, Molt 16, Jurkat, Daudi, UT7,
CEM, Molt4, and CMK (Cheng and H a a ~ , 3Sugimoto
~
et
al,” and our unpublished observations), implicating that inactivation of multiple tumor-suppressor genes may occur
during development of human leukemias. The frequency of
the homozygous deletion in cell lines were about 40% in
our study and may comparable to 9 of 14 cell lines (64.3%)
in the report by Nobori et aLZ4However, in patients’ samples,
the frequency is relatively low, as is the case with that of
the p53 gene alteration^.^.^^.'^ There could be two possible
explanations for the difference between cell lines and pa-
(t4;11)(q21;q23).
6q-, 9p+, 9p-, 14q-, 14q+,
lp-,
3p-,
7pf.
lop+,
15p+,
15q- 19p+, +mar.
+mar.
-2Op+, +mar.
tients’ samples. One explanation is that leukemic cells with
CDK4I inactivation, more probably by homozygous loss,
take advantage of acquiring immortality (a cause for cell
immortalization), and the other is that, after immortalization,
cell lines may become more prone to deletion of this locus
(a result of cell imm~rtaIization).~~~’
We have shown loss of both alleles of this gene in 4 of
72 (5.6%) samples from patients with acute phase of leukemias and 4 of 14 (28%) of patients with ALL. Our findings
suggest that CDK4I gene inactivation was not merely the
result of establishment of a cell line, but possibly related to
de novo development of human leukemia. The clinical and
cytogenetical findings in these 4 cases were summarized in
Table 3. Although the deletion seems to occur in every cell
lineage in cell lines, all 4 patients with the homozygous loss
were confined to ALL, which may reflect the findings that
loss of 9p21 locus is much more frequently found in ALL?’”
In this context, it is worth noting that inactivation of the
CDK4I gene was detected in 2 cases without cytogenetic
abnormalities in 9p2 l locus, suggesting the actual frequency
of loss of function of this gene may be higher than predicted
by cytogenetic analysis in ALL, which may in turn implicate
the importance of inactivation of this gene in ALL leukemogenesis.
In contrast, the homozygous loss of the CDK4I gene was
not detected in 14 cases with blastic crisis of CML and 45
cases with AML. We may safely conclude that inactivation
of the CDK4I through deletion of both alleles is, if it exists,
rare in AML. However, in this study, we did not examine
the other mechanisms through which the CDK4I become
inactivated, eg, point mutations of this gene. It is well known
that tumor-suppressor genes, including p53 and RB genes,
undergo inactivation by point mutation^."^ The existence of
point mutations of the CDK4I gene were shown at a high
frequency in melanoma cell linesz4and, therefore, this possibility should also be extensively addressed in leukemia patients in further studies.
ACKNOWLEDGMENT
We thank Dr Yasuhide Hayashi (Department of Pediatrics, University of Tokyo, Tokyo, Japan) and Dr T. Shikano (Department of
Pediatrics, University of Hokkaido, Hokkaido, Japan) for providing
samples from patients. We also give thanks to Dr Miyoshi for his
generous gift of an AML 1 probe, C6E3SS2.
REFERENCES
1. Weinberg RA: Tumorsuppressor
gene. Science 2541 138,
1991
2. Hollstein M, Sidransky D, Vogelstein B, Harris CC: p53 mutations in human cancers. Science 253:49, 1991
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
CDK41 ( ~ 1 6 GENE
)
IN HUMAN LEUKEMIAS
3. Levine A J , Momand J, Finlay CA: The p53 tumor suppressor
gene. Nature 351:453, 1991
4. Fearon ER, Vogelstein BA: Genetic model for colorectal tumorigenesis. Cell 61:759, 1990
5. Nigro JM, Baker SJ, Preisinger AC, Jessup JM, Hostetter R,
Cleary K, Bigner SH, Davidson N, Baylin S , Devilee P, Glover T,
Collins FS, Weston A, Modali R, Harris CC, Vogelstein B: Mutations in the p53 gene occur in diverse human tumour types. Nature
342:705, 1989
6. Sugimoto K, Toyoshima H, Sakai R, Miyagawa K, Hagiwara
K, Hirai H, Ishikawa F, Takaku F: Mutations of the p53 gene in
lymphoid leukemia. Blood 77:1153, 1991
7. Chen YC, Chen PJ, Yeh SH, Tien HF, Wang CH, Tang JL,
Hong RL: Deletion of the human retinoblastoma gene in primary
leukemias. Blood 76:2060, 1990
8. Li Y, Bollag G, Clark R, Stevens J, COMOY L,Fults D, Ward
K, Friedman E, Samowitz W, Robertson M: Somatic mutations in
the neurofibromatosis 1 gene in human tumors. Cell 69:275, 1992
9. DeClue JE, Papageorge AG, Fletcher JA, Diehl SR, Ratner N.
Vass WC, Lowy DR: Abnormal regulation of mammalian p2lras
contributes to malignant tumor growth in von Recklinghausen (type
1) neurofibromatosis. Cell 69:265, 1992
10. Call KM, Glaser T, It0 CY, Buckler AJ, Pelletier J, Haber
DA, Rose EA, Kral A, Yeger H, Lewis WH, Jones C, Houseman
DE: Isolation and characterization of a zinc finger polypeptide gene
at the human chromosome 11 Wilms’ tumor locus. Cell 60:509,
1990
11. Gessler M, Poustka A, Cavenee W, Neve RL, Orkin SH,
Bruns CA: Homozygous deletion in Wilms tumours of a zinc-finger
gene identified by chromosome jumping. Nature 343:774, 1990
12. Norbury C, Nurse P Animal cell cycles and their control.
Annu Rev Biochem 61:441, 1992
13. M m J: How cell cycle toward cancer. Science 263:319, 1994
14. Xiong Y, Connolly T, Futcher B, Beach D: Human D-type
cyclin. Cell 65:691, 1991
15. Sherr CJ: Mammalian G1 cyclins. Cell 73:1059, 1993
16. Nigg EA: Targets of cyclin-dependent protein kinases. Cum
Opin Cell Biol 5:187, 1993
17. Matsushime H, Ewen ME, Strom DK, Kat0 JY, Hanks SK,
Roussel MF, Sherr CJ: Identification and properties of an atypical
catalytic subunit (p34PSK-J3/cdk4) for mammalian D type G1
cyclins. Cell 71:323, 1992
18. Xiong Y, Zhang H, Beach D: D type cyclins associate with
multiple protein kinases and the DNA replication and repair factor
PCNA. Cell 71505, 1992
19. Kat0 J, Matsushime H, Hiebert SW, Ewen ME, Sherr CJ:
Direct binding of cyclin D to the retinoblastoma gene product (pRb)
and pRb phosphorylation by the cyclin D-dependent kinase CDK4.
Genes Dev 7:331, 1993
20. Ewen ME, Sluss HK, Shen CJ, Matsushime H, Kat0 J, Livingston DM: Functional interactions of the retinoblastoma protein
with mammalian D-type cyclins. Cell 73:487, 1993
21. Xiong Y, Hannon GJ, B a n g H, Casso D, Kobayashi R, Beach
D: p21 is a universal inhibitor of cyclin kinases. Nature 366:701,
1993
22. Xiong Y, Zhang H, Beach D: Subunit rearrangement of the
2435
cyclin-dependent kinases is associated with cellular transformation.
Genes Dev 7:1572, 1993
23. Serrano M, Hannon GJ, Beach DA: New regulatory motif in
cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature 366:704, 1993
24. Nobori T, Miura K, Wu DJ, Lois A, Takabayashi K, Carson
DA: Deletion of the cyclin-dependent kinase-4 inhibitor gene in
multiple human cancers. Nature 368:753, 1994
25. Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman
K, Tavtigian SV, Stockert E, Day RS ID, Johnson BE, Skolnick
MH: A cell cycle regulator potentially involved in genesis of many
tumor types. Science 264:436, 1994
26. Chornczynski P, Sacchi N: Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162:156, 1987
27. Kowalczyk J, Sandberg AA: A possible subgroup ofALL
with 9p-. Cancer Genet Cytogenet 9:383, 1983
28. Chilcote RR, Brown E, Rowley J: Lymphoblastic leukemia
with lymphomatous features associated with abnormalities of the
short arm of chromosome 9. N Engl J Med 313:286, 1985
29. Pollak C, Hagemeijer A: Abnormalities of the short arm of
chromosome 9 with partial loss of material in hematological disorders. Leukemia 1541, 1987
30. Diaz MO, Rubin CM, Harden A, Ziemin S , Larson RA, Le
Beau MM, Rowley JD: Deletions of interferon genes in acute lymphoblastic leukemia. N Engl J Med 322:77, 1990
31. Fountain JW, Karayiorgou M, Emstoff MS, Kirkwood JM,
Vlock DR. Titus Ernstoff L, Bouchard B, Vijayasaradhi S , Houghton
AN, Lahti J, Housman DE, Dracopoli NC: Homozygous deletions
within human chromosome band 9p21 in melanoma. Roc Natl Acad
Sci USA 89:10557, 1992
32. Petty EM, Gibson LH, Breg WR, Bums JP, Yang Feng TI:
Mosaic dup (9p) diagnosed by fluorescence in situ hybridization
(FISH). Am J Med Genet 45770, 1993
33. Cheng J, Hass M: Frequent mutations in the p53 tumor suppressor gene in human leukemia T-cell lines. Mol Cell Biol 10:5502,
1990
34. Sugimoto K, Toyoshima H, Sakai R, Miyagawa K, Hagiwara
K, Ishikawa F, Takaku F, Yazaki Y, Hirai H: Frequent mutations
in the p53 gene in human myeloid leukemia cell lines. Blood
79:2378, 1992
35. Sugimoto K, Naoto H, Hideo T, Shygeru C, Hiroyuki M,
Fumimaro T, Yoshio Y, Hisamaru H: Mutations of the p53 gene in
myelodysplastic syndrome (MDS) and MDS-derived leukemia.
Blood 81:3022, 1993
36. Bischoff FZ, Yim SO, Pathak S , Grant G, Siciliano M, Giovanella BC, Strong LC, Tainsky MA: Spontaneous abnormalities in
normal fibroblasts from patients with Li-Fraumeni cancer syndrome:
aneuploidy and immortalization. Cancer Res 50:7979, 1990
37. Sidransky D, Mikkelsen T, Schwechheimer K, Rosenblum
ML, Cavanee W, Vogelstein B: Clonal expansion of p53 mutant
cells is associated with brain tumour progression. Nature 355:846,
1992
38. Yin Y, Tainsky MA, Bischoff F Z , Strong LC, Wahl GM:
Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 70:937, 1992
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1994 84: 2431-2435
Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16)
gene in human leukemias
S Ogawa, N Hirano, N Sato, T Takahashi, A Hangaishi, K Tanaka, M Kurokawa, T Tanaka, K
Mitani and Y Yazaki
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