Clinical Cancer Research Folkman Stand Up to Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, C. T.
Right arrow Articles by Cordon-Cardo, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, C. T.
Right arrow Articles by Cordon-Cardo, C.
Clinical Cancer Research Vol. 5, 977-983, May 1999
© 1999 American Association for Cancer Research

Advances in Brief

Overexpression of the Cyclin-dependent Kinase Inhibitor p16 Is Associated with Tumor Recurrence in Human Prostate Cancer1

Cheryl T. Lee, Paola Capodieci, Iman Osman, Melissa Fazzari, Joseph Ferrara, Howard I. Scher and Carlos Cordon-Cardo2

Urology Service, Department of Surgery [C. T. L.]; Department of Pathology [P. C., C. C-C.]; Genitourinary Service, Department of Medicine [I. O., H. I. S.]; and Department of Biostatistics and Epidemiology [M. F., J. F.], Memorial Sloan-Kettering Cancer Center, New York, New York 10021


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The INK4A gene maps to the 9p21 region and was initially described [M. Serrano et al., Nature (Lond.), 366: 704–707, 1993; A. Kamb et al., Science (Washington DC), 264: 436–440, 1994] as encoding a 148-amino-acid protein termed p16. The p16 protein associates exclusively with Cdk4 and Cdk6, inhibiting their complexation with D-type cyclins and the consequent phosphorylation of pRb. This contributes to cell cycle arrest. The purpose of the present study was to evaluate patterns of p16 expression in a well-characterized cohort of prostatic adenocarcinomas while exploring potential associations between alterations of p16 and clinicopathological variables. Normal and malignant tissues from 88 patients with prostate carcinoma were examined. In situ hybridization and immunohistochemistry assays were used to determine the status of the INK4A exon 1{alpha} transcripts and levels of p16 protein, respectively. Associations between altered patterns of expression and clinicopathological variables, including pretreatment prostate-specific antigen (PSA) level, Gleason grade, pathological stage, and hormonal status, were evaluated using the Mantel-Haenszel {chi}2 test. Biochemical (PSA) relapse after surgery was evaluated using the Kaplan-Meier method and the log-rank test. Levels of p16 expression and INK4A exon 1{alpha} transcripts in normal prostate and benign hyperplastic tissues were undetectable. However, p16 nuclear overexpression was observed in 38 (43%) prostate carcinomas, whereas the remaining 50 (57%) cases showed undetectable p16 levels. Overexpression of p16 protein was found to correlate with increased INK4A exon 1{alpha} transcripts. Moreover, p16 overexpression was associated with a higher pretreatment PSA level (P = 0.018), the use of neoadjuvant androgen ablation (P = 0.001), and a sooner time to PSA relapse after radical prostatectomy (P = 0.002). These data suggest that p16 overexpression is associated with tumor recurrence and a poor clinical course in patients with prostate cancer.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
The INK4A gene maps to the short arm of chromosome 9 (9p21) and was initially described as encoding a protein of Mr 15,845, termed p16 (1 , 2) . The p16 protein forms binary complexes exclusively with Cdk4 and Cdk6, inhibiting their kinase activity and subsequent pRb phosphorylation during the G1 phase of the cell cycle (1 , 3) . Additional complexity results from the presence of a second INK4A product termed p19ARF (4, 5, 6) . The p19ARF protein has recently been shown to interact with mdm2 and to block mdm2-induced p53 degradation and transactivational silencing (7 , 8) . The two products, p16 and p19ARF, share exons 2 and 3 of the INK4A gene, but have distinct promoters and exon 1 units, exon 1{alpha} (p16) and exon 1ß (p19ARF). The INK4A gene is mutated in a wide variety of tumor cell lines and certain primary tumors (2 , 9, 10, 11, 12, 13, 14) . In addition, methylation of the 5' CpG island of the exon 1{alpha} promoter region is a frequent mechanism of p16 inactivation in primary tumors (15 , 16) .

In prostate cancer, the role of INK4A has not been well elucidated, though analyses using microsatellite markers in the vicinity of the INK4A gene have revealed loss of heterozygosity in a subset of primary and metastatic prostate tumors (17) . Unlike other primary tumors, INK4A inactivation, through either deletions, mutations, or promoter methylation, seems to be an infrequent event in prostate cancer (17, 18, 19, 20, 21, 22) . The present study uses immunohistochemical and in situ hybridization assays to examine patterns of p16 expression in a well-characterized cohort of prostate cancer patients treated with radical retropubic prostatectomy. Associations between altered p16 phenotypes and clinicopathological variables were also studied to further define their potential implications in prostate cancer.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Patient Characteristics and Tissues.
A cohort of patients with prostatic adenocarcinoma undergoing radical prostatectomy at the Memorial Sloan-Kettering Cancer Center (MSKCC) from 1990–1991 was retrospectively evaluated. A total of 88 patients had adequate clinical follow-up and available pathological materials. The median age at the time of surgery was 65 years (range, 46–74 years). The median follow-up time was 64.5 months (range, 10–94 months). Formalin-fixed, paraffin-embedded prostate tissues were obtained from the Department of Pathology. Representative H&E-stained sections were examined to evaluate the histopathological characteristics of each tissue section.

Clinicopathological parameters examined included pretreatment PSA,3 and pathological stage (23) and Gleason grade (24) , both determined based on the radical prostatectomy specimen. The hormonal status of the patients was also evaluated. A portion of the cohort (34 patients or 39%) was treated with neoadjuvant androgen ablation and were defined as hormone-treated. Patients who did not receive neoadjuvant therapy were defined as hormone naíve. Additionally, biochemical relapse was examined. Relapse was defined as an elevation in the serum PSA level in a patient who had previously demonstrated an undetectable PSA level postprostatectomy. That is, only patients who had an undetectable PSA level after surgery were included in the cohort because this indicated that the surgical resection was complete and the patient was free of disease. Patients who had PSA relapse were classified as treatment failures with tumor recurrence.

Immunohistochemistry.
An avidin-biotin immunoperoxidase assay was performed on formalin-fixed, paraffin-embedded tissue sections. Deparaffinized sections were treated with 1% H2O2 to block endogenous peroxidase activity. Sections were subsequently immersed in boiling 0.01% citric acid (pH 6.0) in a microwave oven for 15 min to enhance antigen retrieval, allowed to cool, and incubated with 10% normal horse serum (Organon Tecknika Corp, Westchester, PA), to block nonspecific tissue immunoreactivities. A well-characterized antibody to p16 (Ab-1, Oncogene Research Products, Cambridge, MA; 2 µg/ml final concentration) was then incubated overnight at 4°C. Biotinylated horse antimouse IgG antibodies (Vector Laboratories, Inc., Burlingame, CA; 1:25 final dilution) were used as the secondary reagents. This was followed by avidin-biotin immunoperoxidase complexes (1:25, Vector Laboratories, Inc.) for 30 min. Diaminobenzidine was used as the final chromogen, and hematoxylin was used as the nuclear counterstain. Immunoreactivities, assessed in tissue sections from a single representative block in each case, were classified as a continuum of data from undetectable levels (0%) to homogenous staining levels (100%). Data were independently obtained by two observers, with minor inter-observer variability that was resolved by review of the problem cases. Tumors were grouped into two categories defined as follows: (a) Group A (<=5% nuclear immunoreactivity in tumor cells); and (b) Group B (>5% nuclear immunoreactivity in tumor cells).

In Situ Hybridization.
Primers specific for the exon 1{alpha} sequence of the INK4A gene were used to create digoxigenin-labeled probes for in situ hybridization. Probes were cloned into a PCR-Script recombinant plasmid (Stratagene, La Jolla, CA). Plasmid DNA (1 µg) was linearized using BamHI and XhoI. Antisense and sense riboprobes were generated from in vitro transcription of the linearized DNA using T7 and T3 RNA polymerases, respectively. Transcription was sustained for 2 h at 37°C in 1x transcription buffer (Boehringer Mannheim, Indianapolis, IN), 20 units of RNase inhibitor, 10 mmol/liter each ATP, GTP, and CTP, 6.5 mmol/liter UTP, and 3.5 mmol/liter digoxigenin-UTP. Deparaffinized tissue sections were rinsed in water and PBS for 10 min. The slides were digested with Proteinase K (50 µg/ml) for 18 min at 37°C in PBS and postfixed at 4°C in a freshly prepared solution of 4% paraformaldehyde in PBS for 5 min. Prehybridization was done for 30 min at room temperature in 50% formamide and 2x SSC. The hybridization buffer consisted of 50% deionized formamide (v/v), 10% dextran sulfate (50% stock solution), 2x SSC (20x stock solution), 1% SDS (10% stock solution), and 0.25 mg/ml herring sperm DNA (10 mg/ml).

Hybridization was performed overnight at 45°C applying 10 pmol/liter digoxigenin-labeled riboprobe in 50 µl of hybridization buffer per section under a coverslip. The coverslips were removed and the slides were washed twice in prewarmed 2x SSC for 20 min at 42°C, followed by washes in prewarmed 1x SSC and 0.5x SSC, each at 42°C for 20 min. After these washes, the slides were incubated in normal sheep serum diluted in buffer (pH 7.5) and successively in the same buffer with antidigoxigenin-AP antibody (Boehringer Mannheim) at a dilution of 1:500 for 1 h at room temperature. The visualization was accomplished by nitro-blue tetrazolium 5-bromo-4-chloro-3-indoylphosphate. The slides were counterstained with methyl green and mounted.

INK4A exon 1{alpha} transcript levels were examined in a subgroup of 21 cases. Consecutive tissue sections were used to analyze p16 protein by immunohistochemistry and INK4A exon 1{alpha} transcript levels by in situ hybridization. As in the immunohistochemical analysis, tumors were grouped into two categories defined by the absence (<=5% tumor cells with cytoplasmic staining) or presence (>5% tumor cells with cytoplasmic staining) of transcripts.

Statistical Methods.
The statistical analyses of the data from the 88 primary prostate cancer patients were conducted as follows. The response variable, time to PSA relapse, was defined as the time from radical prostatectomy to the time of first detectable PSA measurement. Patients who did not achieve a nonmeasurable PSA after radical prostatectomy were excluded from the analysis. Patients who were still alive at the time of analysis without relapse were censored at the date of last follow-up. The baseline variables examined were PSA measurement at the time of diagnosis, hormone status, Gleason score (hormone naíve patients only), stage of disease, and percent of p16 protein expression.

Associations between p16 expression and different categorical variables (hormone status, tumor grade, tumor stage, and pretreatment PSA levels) were assessed by the Mantel-Haenszel {chi}2 test. Continuous variables, such as pretreatment PSA, not known to follow a particular distribution were also compared between two or more groups using Wilcoxon nonparametric tests.

The Cox proportional hazards model was used to examine the multivariate relationship between PSA relapse-free time from prostatectomy and the baseline variables listed above. The final model was determined using the "all subsets" procedure in SAS PHREG and the Score criterion (25) . As normal and benign tissues showed little to no p16 expression, positive expression was described as >5% nuclear expression. This cutpoint was specified a priori and used for the subsequent statistical analysis. Immunohistochemical and in situ hybridization studies were completed, analyzed, and recorded blind to clinical information. Kaplan-Meier estimates of relapse-free survival stratified by p16 classification were evaluated. The LIFETEST procedure in SAS was used to generate the Kaplan-Meier estimates and the resulting survival curves (25 , 26) . The log-rank test was used to test the hypothesis of no-survival differences between p16-positive and p16-negative populations.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
The normal human prostate displayed undetectable levels of p16 protein and INK4A exon 1{alpha} transcripts in ductal and acinar epithelial cells. A lack of p16 immunoreactivity in these cells was observed in hormone-treated and hormone-naive cases. Fibromuscular stroma cells also showed undetectable exon 1{alpha} transcript levels. A similar negative pattern of p16 expression was observed on the examination of prostatic tissue affected with benign hyperplasia (Fig. 1)Citation .



View larger version (73K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Immunohistochemistry and in situ hybridization of human benign prostatic hyperplasia (BPH). Consecutive sections of benign hyperplastic tissue were processed as follows: A, immunohistochemical staining with an anti-p16 specific monoclonal antibody reveals undetectable protein levels in both epithelial and stromal cells; B, in situ hybridization shows undetectable INK4A exon 1{alpha} transcripts in both epithelial and stromal cells (antisense probe).

 
To determine the frequency and potential clinical implications of p16 alterations in prostate cancer, we analyzed a cohort of 88 primary prostate carcinomas. Two patterns of p16 protein expression were noted. We observed that 50 (57%) of the 88 cases had very low (<=5% nuclear immunoreactivity; 7 cases) or undetectable (43 cases) levels of p16 protein expression (Group A; Fig. 2ACitation ). In a subgroup of these cases, we also performed in situ hybridization assays, which revealed that all of the cases had undetectable INK4A exon 1{alpha} transcripts (Fig. 2B)Citation . However, we noted that 38 (43%) of the 88 cases displayed nuclear staining with anti-p16 specific antibodies (Group B; Fig. 2CCitation ). Immunoreactivities in tumor cells were further stratified into three categories: (a) 6–29% nuclear staining (n = 11 cases); (b) 30–59% nuclear staining (n = 15 cases); and (c) 60–100% nuclear staining (n = 12 cases). In a subset of these patients, we also conducted in situ hybridization assays with the INK4A exon 1{alpha}-specific probe. All of the cases displaying positive immunoreactivities also displayed moderate-to-high levels of exon 1{alpha} transcripts (Fig. 2D)Citation .



View larger version (141K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Immunohistochemistry and in situ hybridization of human primary prostatic carcinomas. Consecutive sections of primary human prostate cancer tissues were processed as follows: A, immunohistochemical staining with an anti-p16 monoclonal antibody shows lack of immunoreaction in tumor cells and stromal elements; B, in situ hybridization reveals undetectable INK4A exon 1{alpha} transcripts in both tumor and normal stromal elements (antisense probe); C, immunohistochemical staining with an anti-p16 monoclonal antibody shows strong immunoreaction in the nuclei of tumor cells, as well as moderate cytoplasmic staining; and D, in situ hybridization shows high levels of INK4A exon 1{alpha} transcripts in tumor cells when the antisense probe is used. Normal glands (arrow) serve as an internal negative control in both immunohistochemical (C) and in situ hybridization (D) assays.

 
Table 1Citation summarizes the associations between p16 phenotypes and clinicopathological variables that were assessed by {chi}2 analyses. Immunohistochemical detection of p16 was not associated with Gleason grade, described as either low (Gleason grade 4- 6) or high (Gleason grade 7–10; P = 0.153). Similarly, no association was observed between p16 nuclear expression and pathological stage, defined as organ-confined (T1, T2) and non-organ-confined (T3, T4, or lymph node+; P = 0.087). However, there was a strong association between p16 nuclear expression and pretreatment PSA levels, based on cutoff points of <4, 4–10, and >10 ng/ml (P = 0.018). A similar result was observed when PSA was assessed as a continuous variable (P = 0.01). In addition, we noted a significant correlation between p16 nuclear expression and the use of neoadjuvant androgen ablation (P = 0.001).


View this table:
[in this window]
[in a new window]

  		Table 1 Association of p16 immunoreactivity with tumor grade, hormonal status, tumor stage, and preoperative PSA levels

 
A strong association was also found between p16 nuclear overexpression and tumor recurrence, as defined by biochemical (PSA) relapse. Increasing p16 expression correlated with an increased relative hazard of relapse, which suggested a continuous relationship of the data. Overall, tumor recurrence was observed in 34 (39%) of 88 cases. Thirteen (26%) of 50 cases with undetectable-to-low p16 expression (Group A) developed tumor recurrence. However, tumor recurrence was observed in 21 (55%) of 38 cases with p16 nuclear overexpression (Group B; P = 0.002; Fig. 3Citation ). Nevertheless, in a multivariate analysis adjusted for tumor grade, pretreatment PSA, and pathological stage, overexpression of p16 did not contribute prognostic information over pretreatment PSA, the strongest independent predictor of tumor recurrence.



View larger version (15K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Kaplan-Meier curves using the log-rank test for postprostatectomy recurrence-free survival of patients with primary prostate cancer (n = 88) stratified by p16 expression (Group A or Group B). Patients with PSA relapse were classified as failures, whereas patients without evidence of disease or dead from other or unknown causes were censored. Median time to relapse was 46.25 months for Group B and has not been reached for Group A.

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
Normal prostate tissues display undetectable levels of p16 protein and INK4A exon 1{alpha} transcripts. It has been reported that p16 expression is low to undetectable in most normal human tissues analyzed (15 , 27 , 28) . In support of these observations, there are relatively low and near-constant levels of p16 protein and mRNA throughout the cell cycle of normal lymphocytes in culture (29) . The lack of p16 expression in hyperplastic glands parallels that of normal prostatic tissue. On the basis of these data, it is our hypothesis that these negative p16 phenotypes reflect basal physiological levels of p16.

Primary prostatic adenocarcinomas revealed two distinct p16 phenotypes. Most tumors (57% of cases) were found to have undetectable or very low levels of p16 protein expression (Group A). This was associated with low levels or absence of INK4A exon 1{alpha} transcripts. Another group of tumors (43%) showed elevated p16 protein expression (Group B) that was consistently associated with increased INK4A exon 1{alpha} transcripts. These findings suggest an up-regulation of the INK4A-{alpha} gene, resulting in p16 protein overexpression. Patients in Group B had a more aggressive course, demonstrated by high levels of pretreatment PSA (P = 0.018) and a sooner time to biochemical (PSA) relapse (P = 0.002). A worse prognosis for Group B is also revealed by the trending association of p16 overexpression with higher pathological stage.

The negative phenotype observed in Group A may correspond to the normal physiological state, reflecting low-to-undetectable p16 levels. Alternatively, it could be related to mutations affecting the INK4A gene, especially homozygous deletions, or methylation of the INK4A exon 1{alpha} promoter region. Nevertheless, it has been reported that these events are infrequent in prostate cancer (17, 18, 19, 20, 21, 22) . Furthermore, it seems that tumors with INK4A mutations have a more aggressive clinical course (30, 31, 32) . Contrary to this, in the present study, we observed that Group A patients had a less aggressive behavior than Group B patients. For these reasons, we hypothesize that the negative phenotype observed in Group A is more likely a reflection of the normal physiological state.

The up-regulation of the INK4A-{alpha} gene, resulting in the overexpression of p16 protein, may develop through different mechanisms. An association between increased p16 transcript and protein levels occurs in tumor cell lines and certain primary neoplasms that lack functional pRb (1 , 33, 34, 35, 36) . Moreover, p16-mediated inhibition of cell cycle progression seems to be dependent on functional pRb (37 , 38) . These data support an association between p16 and pRb, in which the absence of functional pRb limits p16 activity and possibly promotes INK4A-{alpha} up-regulation. Alternatively, enhanced activation of the INK4A-{alpha} gene may occur. E2F1, a direct activator of the INK4A exon 1ß promoter, does not seem to directly activate INK4A-{alpha} transcription (39) . However, evidence does exist for an indirect effect; E2F1 overexpression has been reported to markedly increase p16 transcripts and p16-related cyclin-dependent kinase inhibitor activity (40) . Overexpression of cyclin D1 and/or of Cdk4 may also influence p16 expression through a compensatory feedback loop in which deregulation of cyclin D/Cdk4 complexes results in increased levels of p16 protein (27 , 41) . In summary, it seems that an altered RB axis could trigger p16 overexpression in certain systems.

Cellular stress produced by replicative senescence (42, 43, 44) , hyperthermia (45) , and UV irradiation (46) has been reported to trigger p16 overexpression. In the present study, another type of cellular stress—androgen ablation—may account in part for this observed phenomenon. A subset of patients were treated with neoadjuvant androgen ablation, a strategy reported to decrease the incidence of positive surgical margins after prostatectomy (47) . In the present study, p16 overexpression was observed in 71% of hormone-treated versus 26% of hormone-naíve patients (P = 0.001). These data suggest that p16 expression may be enhanced by androgen depletion. Androgens are known to modulate the expression of other cyclin-dependent kinase inhibitors such as p27 and p21 (48) . In addition, it has been reported that the presence of androgens triggers down-regulation of p16 in LNCaP cells (49) , a finding consistent with our observation of p16 overexpression in cases of androgen ablation. It is also possible that the association between p16 expression and androgen ablation may, in part, reflect staging bias by clinicians. In this setting, patients thought to have advanced disease may have been treated with neoadjuvant therapy.

On the basis of the above referred data, it is our working hypothesis that p16 overexpression in prostate cancer represents an altered phenotype that identifies a subgroup of patients with a higher likelihood of postsurgical failure and tumor recurrence. In support of this postulate, a preliminary report in prostate cancer has demonstrated an association between p16 overexpression and poor outcome as related to biochemical failure (50) . In addition, p16 overexpression has been associated with tumor progression and a poor prognosis in ovarian (51) and breast cancers (36) . Although p16 acts as a negative cell cycle regulator, specific mechanisms may contribute to its altered expression, overcoming p16-mediated tumor suppressor activities. Ongoing studies may elucidate mechanisms of p16 overexpression relative to androgen depletion and/or alterations in the RB axis.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by National Cancer Institute Grant CA-DK-47650 (C. C-C.) and the PepsiCo Foundation (H. I. S.). C. L. is a Urological Oncology Fellow recipient of a Fellowship from National Cancer Institute Training Program CA09501. Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021. Phone: (212) 639-7746; Fax: (212) 794-3186; E-mail: cordon-c{at}mskcc.org Back

3 The abbreviation used is: PSA, prostate-specific antigen. Back

Received 12/16/98; revised 1/18/99; accepted 1/25/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

  1. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell cycle control causing specific inhibition of cyclinD-cdk4. Nature (Lond.), 366: 704-707, 1993.[Medline]
  2. Kamb A., Gruis N., Weaver-Feldhaus J., Liu Q., Harshman K., Tavtigian S., Stockert E., Day R., III, Johnson B., Skolnick M. A cell cycle regulator potentially involved in genesis of many tumor types. Science (Washington DC), 264: 436-440, 1994.[Abstract/Free Full Text]
  3. Quelle D., Ashmun R., Hannon G., Rehberger P., Trono D., Richter H., Walker C., Beach D., Sherr C., Serrano M. Cloning and characterization of murine p16INK4a and p15INK4b genes. Oncogene, 11: 635-645, 1995.[Medline]
  4. Quelle D., Zindy F., Ashmun R., Sherr C. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell, 83: 993-1000, 1995.[Medline]
  5. Mao L., Merlo A., Bedi G., Shapiro G., Edwards C., Rollins B., Sidransky D. A novel p16INK4A transcript. Cancer Res., 55: 2995-2997, 1995.[Abstract/Free Full Text]
  6. Stone S., Jiang P., Dayananth P., Tavtigian S., Katcher H., Parry D., Peters G., Kamb A. Complex structure and regulation of the P16 (MTS1) locus. Cancer Res., 55: 2988-2994, 1995.[Abstract/Free Full Text]
  7. Pomerantz J., Schreiber-Agus N., Liegeois N. J., Silverman A., Alland L., Chin L., Potes J., Chen K., Orlow I., Lee H. W., Cordon-Cardo C., DePinho R. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2’s inhibition of p53. Cell, 92: 713-723, 1998.[Medline]
  8. Zhang Y., Xiong Y., Yarbrough W. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell, 92: 725-734, 1998.[Medline]
  9. Jen J., Harper J., Bigner S., Bigner D., Papadopoulos N., Markowitz S., Willson J., Kinzler K., Vogelstein B. Deletion of p16 and p15 genes in brain tumors. Cancer Res., 54: 6353-6358, 1994.[Abstract/Free Full Text]
  10. Spruck C., III, Gonzalez-Zulueta M., Shibata A., Simoneau A., Lin M., Gonzales F., Tsai Y., Jones P. p16 gene in uncultured tumours. Nature (Lond.), 370: 183-184, 1994.[Medline]
  11. Orlow I., Lacombe L., Hannon G., Serrano M., Pellicer I., Dalbagni G., Reuter V., Zhang Z., Beach D., Cordon-Cardo C. Deletion of the p16 and p15 genes in human bladder tumors. J. Natl. Cancer Inst., 87: 1524-1529, 1995.[Abstract/Free Full Text]
  12. Reed A., Califano J., Cairns P., Westra W., Jones R., Koch W., Ahrendt S., Eby Y., Sewell D., Nawroz H., Bartek J., Sidransky D. High frequency of p16 (CDKN2/MTS1/INK4A) inactivation in head and neck squamous cell carcinoma. Cancer Res., 56: 3630-3633, 1996.[Abstract/Free Full Text]
  13. Washimi O., Nagatake M., Osada H., Ueda R., Koshikawa T., Seki T., Takahashi To., Takahashi Ta. In vivo occurrence of p16 (MTS1) and p15 (MTS2) alterations preferentially in non-small cell lung cancers. Cancer Res., 55: 514-517, 1995.[Abstract/Free Full Text]
  14. Takeuchi S., Bartram C., Seriu T., Miller C., Tobler A., Janssen J., Reiter A., Ludwig W., Zimmermann M., Schwaller J., Lee E., Miyoshi I., Koeffler H. P. Analysis of a family of cyclin-dependent kinase inhibitors: p15/MTS2/INK4B, p16/MTS1/INK4A, and p18 genes in acute lymphoblastic leukemia of childhood. Blood, 86: 755-760, 1995.[Abstract/Free Full Text]
  15. Gonzalez-Zulueta M., Bender C. M., Yang A. S., Nguyen T., Beart R. W., Van Tornout J. M., Jones P. A. Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res., 55: 4531-4535, 1995.[Abstract/Free Full Text]
  16. Merlo A., Herman J., Mao L., Lee D., Gabrielson E., Burger P., Baylin S., Sidransky D. 5' CpG island methylation is associated with transcriptional silencing of the tumor suppressor p16/CDKN2/Mts1 in human cancers. Nat. Med., 1: 686-692, 1995.[Medline]
  17. Jarrard D., Bova S., Ewing C., Pin S., Nguyen S., Baylin S., Cairns P., Sidransky D., Herman J., Isaacs W. Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer. Genes Chromosomes Cancer, 19: 90-96, 1997.[Medline]
  18. Mangold K., Takahashi H., Brandigi C., Wada T., Wakui S., Furusato M., Boyd J., Chandler F., Allsbrook W. p16 (CDKN2/MTS1) gene deletions are rare in prostatic carcinomas in the United States and Japan. J. Urol., 157: 1117-1120, 1997.[Medline]
  19. Park D., Wilczynski S., Pham E., Miller C., Koeffler H. P. Molecular analysis of the INK4 family of genes in prostate carcinomas. J. Urol., 157: 1995-1999, 1997.[Medline]
  20. Tamimi Y., Bringuier P., Smit F., van Bokhoven A., Debruyne F., Schalken J. p16 mutations/deletions are not frequent events in prostate cancer. Br. J. Cancer, 74: 120-122, 1996.[Medline]
  21. Chen W., Weghorst M., Sabourin C., Wang Y., Wang D., Bostwick D., Stoner G. Absence of p16/MTS1 gene mutations in human prostate cancer. Carcinogenesis (Lond.), 17: 2603-2607, 1996.[Abstract/Free Full Text]
  22. Gu K., Mes-Masson A. M., Gauthier J., Saad F. Analysis of the p16 tumor suppressor gene in early-stage prostate cancer. Mol. Carcinog., 21: 164-170, 1998.[Medline]
  23. Schroder F., Hermanek P., Denis L., Fair W., Gospodarowicz M., Pavone-Macaluso M. The TNM classification of prostate cancer. Prostate, 4: 129-138, 1992.
  24. Gleason D., Mellinger G. Prediction of prognosis for prostatic adenocarcinoma by combined histological grading and clinical staging. J. Urol., 111: 58-64, 1974.[Medline]
  25. SAS Institute Inc. SAS/STAT User Guide, Version 6. Cary, NC: SAS Institute Inc., 1990.
  26. Kaplan E. L., Meier P. Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc., 53: 457-481, 1958.
  27. Yao J., Pollock R. E., Lang A., Tan M., Pisters P. W. T., Goodrich D., El-Naggar A., Yu D. Infrequent mutation of the p16/MTS1 gene and overexpression of cyclin-dependent kinase 4 in human primary soft-tissue sarcoma. Clin. Cancer Res., 4: 1065-1070, 1998.[Abstract]
  28. Shapiro G. I., Edwards C. D., Kobzik L., Lodleski J., Richards W., Sugarbaker D. J., Rollins B. J. Reciprocal Rb inactivation and p16INK4 expression in primary lung cancers and cell lines. Cancer Res., 55: 505-509, 1995.[Abstract/Free Full Text]
  29. Soucek T., Pusch O., Hengstschlager-Ottnad E., Wawra E., Bernaschek G., Hengstschlager M. Expression of the cyclin-dependent kinase inhibitor p16 during the ongoing cell cycle. FEBS Lett., 373: 164-169, 1995.[Medline]
  30. Taga S., Osaki T., Ohgami A., Imoto H., Yoshimatsu T., Yoshino I., Yano K., Nakanishi R., Ichiyoshi Y., Yasumoto K. Prognostic value of the immunohistochemical detection of p16INK4 expression in nonsmall cell lung carcinoma. Cancer (Phila.), 80: 389-395, 1997.[Medline]
  31. Garcia-Sanz R., Gonzalez M., Vargas M., Chillon M., Balanzategui A., Barbon M., Flores M., San Miguel J. Deletions and rearrangements of cyclin-dependent kinase 4 inhibitor gene p16 are associated with poor prognosis in B cell non-Hodgkin’s lymphomas. Leukemia (Baltimore), 11: 1915-1920, 1997.[Medline]
  32. Straume O., Akslen L. Alterations and prognostic significance of p16 and p53 protein expression in subgroups of cutaneous melanoma. Int. J. Cancer, 74: 535-539, 1997.[Medline]
  33. Parry D., Bates S., Mann D. J., Peters G. Lack of cyclin D-cdk complexes in Rb-negative cells correlates with high levels of p16INK4/MTS1 tumour suppressor gene product. EMBO J., 14: 503-511, 1995.[Medline]
  34. Ueki K., Ono Y., Henson J. W., Efird J. T., von Deimling A., Louis D. N. CDKN2/p16 or RB alterations occur in the majority of glioblastomas and are inversely correlated. Cancer Res., 56: 150-153, 1996.[Abstract/Free Full Text]
  35. Li Y., Nichols M. A., Shay J. W., Xiong Y. Transcriptional repression of the D-type cyclin-dependent kinase inhibitor p16 by the retinoblastoma susceptibility gene product pRb. Cancer Res., 54: 6078-6082, 1994.[Abstract/Free Full Text]
  36. Dublin E., Patel N., Gillett C., Smith P., Peters G., Barnes D. Retinoblastoma and p16 proteins in mammary carcinoma: their relationship to cyclin D1 and histopathological parameters. Int. J. Cancer, 79: 71-75, 1998.[Medline]
  37. Lukas J., Parry D., Aagaard L., Mann D. J., Bartkova J., Strauss M., Peters G., Bartek J. Retinoblastoma-protein-dependent cell-cycle inhibition by the tumour suppressor p16. Nature (Lond.), 375: 503-506, 1995.[Medline]
  38. Craig C., Kim M., Ohri E., Wersto R., Katayose D., Li Z., Choi Y., Mudahar B., Srivastava S., Seth P., Cowan K. Effects of adenovirus-mediated p16INK4A expression on cell cycle arrest are determined by endogenous p16 and Rb status in human cancer cells. Oncogene, 16: 265-272, 1998.[Medline]
  39. Robertson K., Jones P. The human ARF cell cycle regulatory gene promoter is a CpG island which can be silenced by DNA methylation and down-regulated by wild-type p53. Mol. Cell. Biol., 18: 6457-6473, 1998.[Abstract/Free Full Text]
  40. Khleif S., DeGregori J., Yee C., Otterson G., Kaye F., Nevins J., Howley P. Inhibition of cyclin D-CDK4/CDK6 activity is associated with an E2F-mediated induction of cyclin kinase inhibitor activity. Proc. Natl. Acad. Sci. USA, 93: 4350-4354, 1996.[Abstract/Free Full Text]
  41. Burns K., Ueki K., Jhung S., Koh J., Louis D. Molecular genetic correlates of p16 cdk4, and pRb immunohistochemistry in glioblastomas. J. Neuropathol. & Exp. Neurol., 57: 122-130, 1998.[Medline]
  42. Hara E., Smith R., Parry D., Tahara H., Stone S., Peters G. Regulation of p16CDKN2 expression and its implications for cell immortalization and senescence. Mol. Cell. Biol., 16: 859-867, 1996.[Abstract]
  43. Palmero I., McConnell B., Parry D., Brookes S., Hara E., Bates S., Jat P., Peters G. Accumulation of p16INK4a in mouse fibroblasts as a function of replicative senescence and not of retinoblastoma gene status. Oncogene, 15: 495-503, 1997.[Medline]
  44. Yeager T., DeVries S., Jarrard D., Kao C., Nakada S., Moon T., Bruskewitz R., Stadler W., Meisner L., Gilchrist K., Newton M., Waldman F., Reznikoff C. Overcoming cellular senescence in human cancer pathogenesis. Genes Dev., 12: 163-174, 1998.[Abstract/Free Full Text]
  45. Valenzuela M. T., Nunez M. I., Villalobos M., Siles E., McMillan T., Pedraza V., Almodovar R. A comparison of p53 and p16 expression in human tumor cells treated with hyperthermia or ionizing radiation. Int. J. Cancer, 72: 307-312, 1997.[Medline]
  46. Wang X. Q., Gabrielli B. G., Milligan A., Dickinson J. L., Antalis T. M., Ellem K. A. O. Accumulation of p16CDKN2A in response to ultraviolet irradiation correlates with a late S-G2-phase cell cycle delay. Cancer Res., 56: 2510-2514, 1996.[Abstract/Free Full Text]
  47. Witjes W., Schulman C., Debruyne F. Preliminary results of a prospective randomized study comparing radical prostatectomy versus radical prostatectomy associated with neoadjuvant hormonal combination therapy in T2–3 N0 M0 prostatic carcinoma. The European Study Group on Neoadjuvant Treatment of Prostate Cancer. Urology, 49 (Suppl.): 65s-69s, 1997.
  48. Kokontis J., Hay N., Liao S. Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest. Mol. Endocrinol., 12: 941-953, 1998.[Abstract/Free Full Text]
  49. Lu S., Tsai S. Y., Tsai M-J. Regulation of androgen-dependent prostatic cancer cell growth: androgen regulation of CDK2, CDK4, and CKI p16 genes. Cancer Res., 57: 4511-4516, 1997.[Abstract/Free Full Text]
  50. Halvorsen O. J., Hostmark J., Haukass , Hoisaeter P., Akslen L. Prognostic importance of p16 and CDK4 proteins in localized prostate cancer. Proc. Am. Assoc. Cancer Res., 38: 526 1997.
  51. Dong Y., Walsh M., McGuckin M., Gabrielli B., Cummings M., Wright R. G., Hurst T., Khoo S. K., Parsons P. Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16ink4A in ovarian cancer is associated with progression and unfavourable prognosis. Int. J. Cancer, 74: 57-63, 1997.[Medline]



This article has been cited by other articles:


Home page
 J. Clin. Pathol.Home page
R Natrajan, W Warren, B Messahel, J S Reis-Filho, M-A Brundler, J S Dome, P E Grundy, G Vujanic, K Pritchard-Jones, and C Jones
Complex patterns of chromosome 9 alterations including the p16INK4a locus in Wilms tumours
J. Clin. Pathol., January 1, 2008; 61(1): 95 - 102.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
B. Shuch, M. Mikhail, J. Satagopan, P. Lee, H. Yee, C. Chang, C. Cordon-Cardo, S. S. Taneja, and I. Osman
Racial Disparity of Epidermal Growth Factor Receptor Expression in Prostate Cancer
J. Clin. Oncol., December 1, 2004; 22(23): 4725 - 4729.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
L. A. Maddison, B. W. Sutherland, R. J. Barrios, and N. M. Greenberg
Conditional Deletion of Rb Causes Early Stage Prostate Cancer
Cancer Res., September 1, 2004; 64(17): 6018 - 6025.
[Abstract] [Full Text] [PDF]

Home page
 JNCI J Natl Cancer InstHome page
C. Gurrieri, P. Capodieci, R. Bernardi, P. P. Scaglioni, K. Nafa, L. J. Rush, D. A. Verbel, C. Cordon-Cardo, and P. P. Pandolfi
Loss of the Tumor Suppressor PML in Human Cancers of Multiple Histologic Origins
J Natl Cancer Inst, February 18, 2004; 96(4): 269 - 279.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
A. Latil, I. Bieche, L. Chene, I. Laurendeau, P. Berthon, O. Cussenot, and M. Vidaud
Gene Expression Profiling in Clinically Localized Prostate Cancer: A Four-Gene Expression Model Predicts Clinical Behavior
Clin. Cancer Res., November 15, 2003; 9(15): 5477 - 5485.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Drobnjak, J. Melamed, S. Taneja, K. Melzer, R. Wieczorek, B. Levinson, A. Zeleniuch-Jacquotte, D. Polsky, J. Ferrara, R. Perez-Soler, et al.
Altered Expression of p27 and Skp2 Proteins in Prostate Cancer of African-American Patients
Clin. Cancer Res., July 1, 2003; 9(7): 2613 - 2619.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
I. B. Roninson
Tumor Cell Senescence in Cancer Treatment
Cancer Res., June 1, 2003; 63(11): 2705 - 2715.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
M. Omura-Minamisawa, M. B. Diccianni, R. C. Chang, A. Batova, L. J. Bridgeman, J. Schiff, S. L. Cohn, W. B. London, and A. L. Yu
p16/p14ARF Cell Cycle Regulatory Pathways in Primary Neuroblastoma: p16 Expression Is Associated with Advanced Stage Disease
Clin. Cancer Res., November 1, 2001; 7(11): 3481 - 3490.
[Abstract] [Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
S. M. Henshall, D. I. Quinn, C. S. Lee, D. R. Head, D. Golovsky, P. C. Brenner, W. Delprado, P. D. Stricker, J. J. Grygiel, and R. L. Sutherland
Overexpression of the Cell Cycle Inhibitor p16INK4A in High-grade Prostatic Intraepithelial Neoplasia Predicts Early Relapse in Prostate Cancer Patients
Clin. Cancer Res., March 1, 2001; 7(3): 544 - 550.
[Abstract] [Full Text]

Home page
 Am. J. Pathol.Home page
P. Schraml, K. Struckmann, R. Bednar, W. Fu, T. Gasser, K. Wilber, J. Kononen, G. Sauter, M. J. Mihatsch, and H. Moch
CDKN2A Mutation Analysis, Protein Expression, and Deletion Mapping of Chromosome 9p in Conventional Clear-Cell Renal Carcinomas : Evidence for a Second Tumor Suppressor Gene Proximal to CDKN2A
Am. J. Pathol., February 1, 2001; 158(2): 593 - 601.
[Abstract] [Full Text] [PDF]

Home page
 Genes Dev.Home page
C. Abate-Shen and M. M. Shen
Molecular genetics of prostate cancer
Genes & Dev., October 1, 2000; 14(19): 2410 - 2434.
[Full Text]

Home page
 Cell Growth Differ.Home page
A. F. Fribourg, K. E. Knudsen, M. W. Strobeck, C. M. Lindhorst, and E. S. Knudsen
Differential Requirements for Ras and the Retinoblastoma Tumor Suppressor Protein in the Androgen Dependence of Prostatic Adenocarcinoma Cells
Cell Growth Differ., July 1, 2000; 11(7): 361 - 372.
[Abstract] [Full Text]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, C. T.
Right arrow Articles by Cordon-Cardo, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, C. T.
Right arrow Articles by Cordon-Cardo, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online