Affiliations of authors: W. G. Yarbrough (Department of Surgery, Division of Otolaryngology/Lineberger Comprehensive Cancer Center), R. A. Buckmire (Department of Surgery, Division of Otolaryngology), M. Bessho (Lineberger Comprehensive Cancer Center), University of North Carolina at Chapel Hill; E. T. Liu, Division of Clinical Sciences, National Cancer Institute, Bethesda, MD.
Correspondence to: Wendell G. Yarbrough, M.D., University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, CB#7295, Chapel Hill, NC 27599-7295 (e-mail: wgy{at}med.unc.edu).
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ABSTRACT |
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INTRODUCTION |
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The amino acid composition suggests that p16 is largely composed of four contiguous ankyrin
repeats. The secondary structure of wild-type p16, as determined by nuclear magnetic resonance,
contains ankyrin repeats that form -helices with interposed turns (8).
The p16 mutant proteins that carried mutations at positions 26, 66, 84, 92, or 124 had nuclear
magnetic resonance spectra that were consistent with the retention of wild-type secondary
structure (9). The tertiary structure of wild-type p16 has a putative cleft
for CDK binding, with key residues thought to be amino acids 46, 47, 50, 79, 84, 88, and 110.
Given the difficulties of assessing the structure of p16 mutants directly, mutants are more frequently analyzed for functional activity. Pollock et al. (10) analyzed 120 point mutations in p16 obtained from cell lines and primary tumors, but the functional importance of the majority of these mutations is not known. To date, familial mutations involved in the pathogenesis of melanoma have received the most attention. These familial p16 mutants tend to cluster in the central portions of the protein and generally result in nonconservative amino acid substitutions. When we compared tumor-associated missense mutations across the ankyrin repeat structures of p16, we found that more than 50% of the mutations in ankyrin repeats I and IV but fewer than 30% of the mutations in ankyrin repeats II and III result in conservative amino acid changes. Likewise, we noted that tumor-associated mutations in the second, third, and early fourth ankyrin repeats occur more frequently at residues that are invariant in all members of the p16 family characterized. Because highly conserved residues in protein families may identify positions critical to protein structure or function, we hypothesized that mutations of these highly conserved residues would be more likely to alter p16 function. Consequently, those highly conserved amino acids in the second, third, and early fourth ankyrin repeats may be critical for p16 function, and tumor-associated mutations elsewhere may not cause functional abnormalities.
To test the biologic and biochemical activities of p16 mutants, we used three assays to assess the ability of the mutants to 1) bind to CDKs, 2) inhibit the activity of CDKs, and 3) arrest cell proliferation. CDK binding is the most commonly used test of p16 function because all inactivating p16 mutations characterized to date result in mutants that lack CDK binding activity (4,5,7,11-14). In this report, we describe the functional activity of 16 naturally occurring p16 mutants carrying mutations that span the entire coding region of p16. From these analyses, we sought to determine whether mutations in regions of the p16 protein that have not been analyzed alter function and to establish whether mutations that alter function cluster within specific structural domains of the p16 protein.
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MATERIALS AND METHODS |
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All p16 mutants analyzed carried single nucleotide substitutions resulting in single amino acid changes that are found in human cancers. We analyzed 16 mutants; 15 mutants were obtained from primary tumors and one mutant (D84V) was obtained from a cell line (UNC7, human squamous cell carcinoma) at passage 2. Mutants carrying point mutations from all four ankyrin repeats of p16 were chosen for analysis. All mutants analyzed, except R87L and D84V, were identified from literature searches (4-6,12,13,15-17). Mutants D84V and R87L were detected by single-strand conformation polymorphism-polymerase chain reaction (SSCP-PCR) with the use of DNA from head and neck squamous cell carcinomas resected at the University of North Carolina at Chapel Hill School of Medicine. Informed written consent from the patient and Institutional Review Board approval were obtained before the tissue was collected and analyzed.
The 20-µL reaction mixture for SSCP-PCR contained 100 ng of genomic DNA, 500 nM of each primer, 1x PCR buffer (The Perkin-Elmer Corp., Foster City, CA; 10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2, and 0.001% gelatin), all four deoxyribonucleoside 5'-triphosphates (each at
1 mM), 5% dimethyl sulfoxide, 2.5 U of AmpliTaq DNA polymerase (Roche
Molecular Biochemicals, Indianapolis, IN), and 100 µCi of deoxycytidine
5'-[-32P]triphosphate. PCR cycling conditions were as
follows: 94 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 30 seconds,
with a final extension at 72 °C for 10 minutes. Exon 1 of p16 was amplified as one fragment,
and exon 2 of p16 was amplified in three segments (2A, 2B, and 2C) with the oligonucleotide
primers described by Hussussian et al. (18). SSCP-PCR products were
diluted 1 : 30 with a solution of 1% sodium dodecyl sulfate (SDS) and 10 mM
EDTA. The diluted product (3 µL) was mixed with an equal volume of stop buffer
(95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and
0.05% xylene cyanol) and denatured at 94 °C for 5 minutes. SSCP reaction products
were subjected to electrophoresis through nondenaturing 6% polyacrylamide gels
containing 5% glycerol, at both room temperature and 4 °C. Negative control samples
containing only wild-type p16 PCR products were included in each gel. PCR products with
altered electrophoretic mobility were further analyzed as follows: The p16 region of interest was
again amplified by PCR. The PCR product was then used as a template for an asymmetric PCR to
produce single forward and reverse DNA strands. The asymmetric PCR was carried out under the
PCR conditions described above but with primers at 1) 500 nM forward primer and 10 nM reverse primer or at 2) 10 nM forward primer and 500 nM reverse
primer. Both forward and reverse single-stranded DNAs were purified and sequenced by the
dideoxynucleotide chain-termination method of Sanger et al. (19) and
were considered to be positive only when both strands had the same mutation. These mutations
were subsequently confirmed by repeating the entire sequencing analysis with separate DNA
samples from the same patients.
Construction and Isolation of Glutathione S-Transferase (GST) Fusion Proteins
A full-length, wild-type p16 complementary DNA (a gift from Dr. Yue Xiong, University of North Carolina at Chapel Hill) containing the coding region homologous to the sequence listed in GenBank (accession numbers U12818, U12819, and U12829) with an additional 24 base pairs of coding sequence at the 5' end was cloned into pGEX-KG. Mutant p16 clones were constructed by use of a site-directed mutagenesis procedure (Altered Sites II), as suggested by the manufacturer (Promega Corp., Madison, WI). Mutations were confirmed by sequencing both DNA strands. CDK6-GST and cyclin D1-GST fusion constructs (in pGEX-KG) were also gifts from Dr. Xiong. A GST-Rb fusion plasmid encoding the carboxyl-terminal 148 amino acids (hereafter referred to as GST-Rb) was constructed as previously described (20). Fusion proteins were expressed in Escherichia coli and purified by passage over glutathione-Sepharose as described previously (7). Samples were concentrated with Centricon-30 spin columns (Amicon, Inc., Beverly MA). The GST-p16 protein concentration was estimated by comparison with protein of known concentration after polyacrylamide gel electrophoresis and Coomassie blue staining.
Binding of CDK4 and CDK6
In vitro transcription and translation of CDK4 and CDK6 were performed with [35S]methionine and T7 TNT in vitro translation kit according to the manufacturer's instructions (Promega Corp.). Wild-type and mutant GST-p16 proteins (500 ng) were incubated with 4 µL of the appropriate in vitro-translated CDK for 30 minutes at 25 °C and 37 °C. Binding of CDK4 and CDK6 by wild-type or mutant GST-p16 species was assessed by affinity precipitation with glutathione-Sepharose (Pharmacia LKB Biotechnology, Piscataway, NJ). The precipitated proteins were washed three times with Nonidet P-40 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Nonidet P-40, and 50 mM NaF), followed by SDS-polyacrylamide gel electrophoresis and autoradiography.
Inhibition of CDK6 and CDK-Activating Complex Activities
The CDK-activating complex was immunoprecipitated with a CDK7 polyclonal antibody from
a BT20 cell lysate (200 µg of total protein) in Nonidet P-40 lysis buffer as described
previously (21). The immunoprecipitated CDK-activating complex was
washed twice in Nonidet P-40 lysis buffer and twice in kinase buffer (50 mM HEPES
[pH 7.3], 10 mM MgCl2, 5 mM MnCl2, and
1 mM dithiothreitol) and resuspended in the same buffer (adjusted to yield 30 µL).
The following proteins then were added sequentially: mutant or wild-type GST-p16 (90 nM, 180 nM, or 360 nM, as described below), 800 ng of GST-CDK6, 800 ng
of GST-cyclin D1, and 600 ng of GST-Rb. Finally, 10 µCi of adenosine
5'-[-32P]triphosphate was added, and the reaction mixture
was incubated at 30 °C for 30 minutes. Reaction products were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography. Mutant or wild-type GST-p16 was
initially analyzed for kinase inhibitory activity at a concentration of 180 nM. The p16
mutant fusion proteins that inhibited CDK6 at this concentration were then assayed at 90 nM. The p16 mutant fusion proteins that did not inhibit CDK6 at 180 nM were then
assayed for kinase inhibitory activity at 360 nM.
Cell Cycle Analysis
U2OS human osteosarcoma cells were cotransfected by electroporation (Gene Pulser with capacitance extender at 350 mV/960 mF; Bio-Rad Laboratories, Hercules, CA) with 1 µg of pCMVCD20 (provided by Dr. Sander van den Heuvel, Massachusetts General Hospital, Boston) and 10 µg of pCI-neo (Promega Corp.) alone or carrying a wild-type or mutant p16 sequence. Growth arrest was similarly assayed with 0.3 µg of pCI-neo alone or carrying wild-type or mutant p16INK4a sequences and 0.3 µg of pCMVCD20. After 48-60 hours, the U2OS cells were stained with a fluorescein isothiocyanate-conjugated anti-CD20 antibody (Becton-Dickinson Immunocytometry Systems, San Jose, CA) as described previously (22). Cells were then fixed and stained with propidium iodide for flow cytometry by use of a Becton-Dickinson FACSort. Data were analyzed with Cell Quest software (Becton-Dickinson Immunocytometry Systems). Propidium iodide intensity of CD20-positive cell populations was used to determine their DNA content and their cell cycle distribution. The data are the average of at least two experiments, in which more than 5000 CD20-positive cells were analyzed per point. Polyacrylamide gel electrophoresis and immunoblotting with p16 antibodies (provided by Dr. Xiong) confirmed expression of wild-type or mutant p16 in cells during cell cycle arrest.
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RESULTS |
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We collected 16 mutations in p16, 14 tumor-associated somatic and
germline missense mutations identified in a literature search and two
mutations (D84G and R87L) that we isolated from head and neck squamous
cell carcinomas. We detected functional defects in nine of the
corresponding mutants and observed normal CDK binding and kinase
inhibitory activity in 10 of these mutants. A graphic representation of
all p16 mutations in relation to the ankyrin repeat structure of p16 is
shown in Fig. 1, A. We functionally analyzed p16
mutants that carried missense mutations that altered amino acids in
each of the ankyrin repeats, as well as in the region carboxyl-terminal
to the fourth ankyrin motif (Fig. 1
, B). The distribution of mutants
analyzed mimicked the distribution of tumor-associated mutations
described in the literature. Of the mutants selected, eight have been
previously characterized to various degrees. We examined these mutants
again because of inconsistencies in the literature derived from the use
of a variety of assays and because of the fact that not all functions
of p16 had been characterized in most mutants. In addition, the
possibility remained that assays of directed kinase inhibition and
growth arrest could detect subtle functional defects that may have been
overlooked with other assay systems.
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Binding of CDK4 and CDK6
The ability of p16 mutant proteins to bind stably to CDK4 and CDK6
was tested by affinity co-precipitation of in vitro-translated
CDK4 or CDK6 with GST-p16 fusion proteins as previously described
(7). The p16 mutants D84V and R87P had no detectable CDK
binding (Fig. 2), and mutant D84G had reduced
binding to in vitro-translated CDK4 and CDK6. All of the other
mutants tested had binding activity equivalent to that of wild-type
p16. A recent report (23) of p16 mutants that had
temperature-sensitive CDK binding activity led us to test CDK4 and CDK6
binding at 37 °C, but we found no p16 mutant with
temperature-conditional binding to CDKs (data not shown).
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The CDK inhibitory activity of the panel of p16 mutants was tested
in an in vitro kinase inhibition assay. Wild-type or mutant
GST-p16 fusion protein at a final concentration of 360 nM, 180
nM, or 90 nM was added to activated complexes of
GST-CDK6 and GST-cyclin D1, and the resulting phosphorylation of a
GST-Rb carboxyl-terminal (Rb-CT) fusion protein was assessed
(7). To determine whether p16 mutants that failed to inhibit
CDK activity at 180 nM retained partial activity, we then
assayed these mutants at 360 nM. Likewise, to determine
whether p16 mutants that inhibited CDK activity fully at 180
nM had more subtle defects, we assayed the mutant at 90
nM. As expected, mutants D84V and R87P, with no detectable
binding to CDKs, did not inhibit the activity of CDK6 at any
concentration (Fig. 3). Mutant D84G, which had decreased
binding to CDKs, also failed to inhibit kinase activity in our standard
assay (180 nM); however, when added at a twofold greater
concentration (360 nM), it partially inhibited the activity.
Of the 13 mutants that retained normal CDK4/6 binding, two (P48L and
R87L) failed to inhibit CDK6 kinase activity at 180 nM but did
inhibit it at 360 nM. Mutant D74N partially inhibited CDK6
activity under the more stringent assay conditions (90 nM),
but all other mutants inhibited equally well at either 180 nM
or 90 nM. In summary, of 16 mutants, six (P48L, D74N, D84G,
D84V, R87L, and R87P) had detectable defects in kinase inhibition. Each
mutant with defective binding of CDKs also had defective kinase inhibition.
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When introduced into cells containing functional Rb protein, wild-type p16 causes growth arrest in the G1 phase of the cell cycle. To determine whether p16 mutant proteins could function similarly, we ectopically expressed all mutants in U2OS cells and then analyzed the cell cycle status of these cells by flow cytometry. The G1-phase arrest caused by the p16 mutants was then compared with the arrest caused by wild-type p16. To standardize results from multiple experiments, we used the magnitude of the G1 arrest after transfection with wild-type p16 as the 100% reference. If no proliferative arrest was observed (i.e., the G1 fraction in mutant p16-transfected cells was equal to the G1 fraction in cells transfected with vector alone), a value of 0% was assigned. Negative numbers indicate that cells transfected with the p16 expression vector had fewer cells in G1 phase than cells transfected with the vector control. At least 5000 transfected cells were analyzed for each data point, and results of multiple experiments were combined to determine the mean and standard deviation. Expression of p16 proteins after transfection was confirmed by gel electrophoresis of a cell lysate followed by immunoblotting with a p16-specific antibody.
A representative experiment showing expression of p16 mutants after transfection with 0.3
µg of expression plasmid is depicted in Fig. 4. The ability of p16
mutants to cause growth arrest in G1 phase was initially analyzed by transfecting cells
with 10 µg of the expression vector carrying p16 sequences. As expected, mutants that had
defective CDK binding (D84G, D84V, and R87P) failed to inhibit growth (Fig. 4
). The mutant that had a decreased ability to inhibit CDK6 kinase activity (P48L) also
did not cause growth arrest in the G1 phase under these conditions. The cell cycle
arrest caused by the remaining 12 mutants was roughly equivalent to that caused by wild-type
p16.
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Thus, all of the p16 mutants containing amino acid alterations in the second or third ankyrin
repeat had defective growth arrest properties. Reciprocally, wild-type p16 and all mutants
carrying a mutations outside ankyrin repeats II and III caused equivalent cell cycle arrests in the G1 phase (Fig. 4).
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DISCUSSION |
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The three p16 mutations that confer defective CDK binding (D84G, D84V, and R87P) were
located at or immediately adjacent to amino acid positions thought to be critical for the direct
interaction with CDKs (9). Our binding assay mimics other assays
commonly described in the literature; however, on the basis of our results, we cannot rule out the
possibility that some nonfunctional mutants have subtle binding defects that would not be detected
with this binding assay. Our functional data are in agreement with and strengthen the structural
data, in that amino acid alterations outside the putative CDK binding cleft did not destabilize the
binding between p16 mutants and CDKs. All p16 mutants with defective CDK binding were
inactive in each of the known functions of p16, confirming that CDK binding is necessary for p16
function. Several mutants retained CDK binding, yet they had other functional defects, suggesting
that CDK binding, although necessary for p16 activity, is insufficient for full inhibition of CDK or
for growth arrest. Mutations outside the CDK binding cleft could theoretically alter p16 function
by decreasing protein stability or subcellular localization. Protein stability, localization, or an as
yet undetermined defect resulting from p16 missense mutations would not be detected by in
vitro assays. Despite the use of identical expression constructs and transfection conditions,
protein levels of p16 mutants showed some variation (Fig. 4). For
instance, mutant H83Y, which had binding and kinase inhibitory functions equivalent to those of
wild-type p16, was consistently expressed at lower levels, possibly as a consequence of decreased
protein stability. Perhaps because of the assay's ability to detect changes in protein stability
or defects dependent on unknown cellular or biochemical functions, we found that the in vivo growth arrest assay was the most sensitive test of p16 activity. As a confirmation of
accuracy, the growth arrest assay detected defects in all mutants that had abnormal CDK binding
or kinase inhibition activity. In addition, the growth arrest assay was able to define functional
defects not detected by the in vitro binding or kinase inhibitory assays. Kinase inhibition
data for one mutant carrying a mutation in ankyrin repeat I and three mutants carrying mutations
in ankyrin repeat IV have been published (9). Although the authors (9) did not test binding or growth arrest, their data suggest that each
mutant tested had decreased kinase inhibitory activity, including the two mutants E26D and
R124H that we found to function normally in all of our assays. The amino acid substitutions in
both proteins are conservative in nature, in that acidic amino acids are replaced with acidic amino
acids and basic amino acids are replaced with basic amino acids. By chance, one of these amino
acid substitutions, E26D, that functioned normally in our battery of assays occurs naturally in
murine p16 (Fig. 1
, C). Although it is possible that a Glu-26 to Asp
substitution could affect functional activities of p16, it is less likely given that Asp-26 occurs
naturally in the murine p16 homologue.
Our analysis does not suggest that all p16 mutations in ankyrin repeats I and IV will confer wild-type activity. Tertiary structure analysis suggests that some residues located early in ankyrin repeat IV may contribute directly to the CDK binding cleft. Support for this model comes from the observation that mutant P114L carrying a mutation in ankyrin repeat IV lacks CDK binding and growth arrest activities (5). Conversely, of nine p16 mutations occurring in ankyrin repeats II and III, mutants resulting from six of these mutations retained partial functional activity. All mutants capable of stable CDK binding could also at least partially inhibit their kinase activity. Likewise, all mutants that could bind and inhibit CDKs in the in vitro assays, excluding P48L, could also partially arrest growing cells. Mutants that retained partial function were found in primary tumors, suggesting that incomplete loss of p16 function may be sufficient for carcinogenesis. However, gene deletion and promoter methylation, both of which totally eliminate expression of the p16 protein, appear to be the methods of p16 gene inactivation most often observed in tumors.
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NOTES |
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We are particularly grateful to Dr. Yue Xiong for his support and reagents. We also thank Dr. Tona Gilmer and her laboratory personnel for assistance with flow cytometric analysis and for helpful discussion.
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REFERENCES |
---|
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---|
1 Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 1995;81:323-30.[Medline]
2 Cairns P, Polascik TJ, Eby Y, Tokino K, Califano J, Merlo A, et al. Frequency of homozygous deletion at p16/CDKN2 in primary human tumours. Nat Genet 1995;11:210-2.[Medline]
3 Herman JG, Merlo A, Mao L, Lapidus RG, Issa JP, Davidson NE, et al. Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common human cancers. Cancer Res 1995;55:4525-30.[Abstract]
4 Ranade K, Hussussian CJ, Sikorski RS, Varmus HE, Goldstein AM, Tucker MA, et al. Mutations associated with familial melanoma impair p16INK4 function [letter]. Nat Genet 1995;10:114-6.[Medline]
5 Koh J, Enders GH, Dynlacht BD, Harlow E. Tumour-derived p16 alleles encoding proteins defective in cell-cycle inhibition. Nature 1995;375:506-10.[Medline]
6
Goldstein AM, Fraser MC, Struewing JP, Hussussian CJ, Ranade
K, Zametkin DP, et al. Increased risk of pancreatic cancer in melanoma-prone kindreds with
p16INK4 mutations. N Engl J Med 1995;333:970-4.
7
Yarbrough WG, Aprelikova O, Pei H, Olshan AF, Liu ET.
Familial tumor syndrome associated with a germline nonfunctional p16INK4a allele. J Natl
Cancer Inst 1996;88:1489-91.
8 Tevelev A, Byeon IJ, Selby T, Ericson K, Kim HJ, Kraynov V, et al. Tumor suppressor p16INK4A: structural characterization of wild-type and mutant proteins by NMR and circular dichroism. Biochemistry 1996;35:9475-87.[Medline]
9 Byeon IJ, Li J, Ericson K, Selby TL, Tevelev A, Kim HJ, et al. Tumor suppressor p16INK4A: determination of solution structure and analyses of its interaction with cyclin-dependent kinase 4. Mol Cell 1998;1:421-31.[Medline]
10 Pollock PM, Pearson JV, Hayward NK. Compilation of somatic mutations of the CDKN2 gene in human cancers: non-random distribution of base substitutions. Genes Chromosomes Cancer 1996;15:77-88.[Medline]
11 Lukas J, Parry D, Aagaard L, Mann DJ, Bartkova J, Strauss M, et al. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature 1995;375:503-6.[Medline]
12 Reymond A, Brent R. p16 proteins from melanoma-prone families are deficient in binding to Cdk4. Oncogene 1995;11:1173-8.[Medline]
13 Yang R, Gombart AF, Serrano M, Koeffler HP. Mutational effects on the p16INK4a tumor suppressor protein. Cancer Res 1995;55:2503-6.[Abstract]
14
Quelle DE, Cheng M, Ashmun RA, Sherr CJ. Cancer-associated
mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative
reading frame protein p19ARF. Proc Natl Acad Sci U S A 1997;94:669-73.
15 Yoshida S, Todoroki T, Ichikawa Y, Hanai S, Suzuki H, Hori M, et al. Mutations of p16Ink4/CDKN2 and p15Ink4B/MTS2 genes in biliary tract cancers. Cancer Res 1995;55:2756-60.[Abstract]
16 Zhang SY, Klein-Szanto AJ, Sauter ER, Shafarenko M, Mitsunaga S, Nobori T, et al. Higher frequency of alterations in the p16/CDKN2 gene in squamous cell carcinoma cell lines than in primary tumors of the head and neck. Cancer Res 1994;54:5050-3.[Abstract]
17 Mori T, Miura K, Aoki T, Nishihira T, Mori S, Nakamura Y. Frequent somatic mutation of the MTS1/CDK4I (multiple tumor suppressor/cyclin-dependent kinase 4 inhibitor) gene in esophageal squamous cell carcinoma. Cancer Res 1994;54:3396-7.[Abstract]
18 Hussussian CJ, Struewing JP, Goldstein AM, Higgins PA, Ally DS, Sheahan MD, et al. Germline p16 mutations in familial melanoma. Nat Genet 1994;8:15-21.[Medline]
19 Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 1977;74:5463-7.[Abstract]
20 Craven RJ, Cance WG, Liu ET. The nuclear tyrosine kinase rak associates with the retinoblastoma protein pRb. Cancer Res 1995;55:3969-72.[Abstract]
21
Aprelikova O, Xiong Y, Liu ET. Both p16 and p21 families of
cyclin-dependent kinase (CDK) inhibitors block the phosphorylation of cyclin-dependent kinases
by the CDK-activating kinase. J Biol Chem 1995;270:18195-7.
22 van den Heuvel S, Harlow E. Distinct roles for cyclin-dependent kinases in cell cycle control. Science 1993;262:2050-4.[Medline]
23 Parry D, Peters G. Temperature-sensitive mutants of p16CDKN2 associated with familial melanoma. Mol Cell Biol 1996;16:3844-52.[Abstract]
Manuscript received March 1, 1999; revised July 12, 1999; accepted July 28, 1999.
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