Suppression of growth in vitro and tumorigenicity in vivo of human carcinoma cell lines by transfected p16INK4

The function of p16^INK4 as a putative tumor suppressor gene was examined by investigating its ability to inhibit the growth of cancer cell lines in vitro and tumor formation in vivo. A p16^INK4 cDNA expression vector was transfected into five human cancer cell lines that varied in their p16^INK4 and retinoblastoma (Rb) status. Suppression of colony-forming efficiency was seen in four cell lines. Of two cell lines wild type for p16^INK4 but null for Rb protein expression, one (Hep 3B) showed inhibition of colony formation, whereas the other (Saos-2) did not. This observation may demonstrate involvement of p16^INK4 independent of Rb. The transfected p16^INK4 gene was frequently selected against and lost during continued growth in vitro. When compared to the colon carcinoma cell line (DLD-1), p16^INK4-transfected DLD-1 clone 1 cells were less tumorigenic in athymic nude mice. Tumors arising from p16^INK4-transfected DLD-1 clones, which were growth suppressed in vitro, either lost the integrated exogenous p16^INK4 or expressed reduced amounts of p16^INK4 protein. Therefore, p16^INK4 was also selected against during tumor formation in vivo. These data are consistent with the hypothesis that p16^INK4 is a tumor suppressor gene. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Infrequent alterations of the p16INK4A gene in liver cancer

We examined the genomic status of the p16^INK4A (inhibitor of cyclin-dependent kinase 4 A) and cyclin-dependent kinase 4 (CDK4) genes in 62 human hepatocellular carcinomas (HCCs), 5 cholangiocellular carcinomas and 6 cell lines derived from human liver cancers. Although no samples showed the homozygous deletion of the p16^INK4A gene, we detected intragenic mutations of the p16^INK4A gene in 3 HCCs and one HCC cell line, which led to an amino-acid substitution or a frameshift. In 2 HCC samples with mis-sense mutations of the p16^INK4A gene, loss of heterozygosity on 9p22 was also detected, suggesting that the loss of function of p16 was induced during hepatocarcinogenesis. On the other hand, amplification or rearrangement of the CDK4 gene was not detected in any samples examined in this study. These results indicated that the mutations or deletions of the p16^INK4A gene are not frequent, but may play a role in a sub-set of human HCC. © 1996 Wiley-Liss, Inc.     

International cancer research grants

Ahderrant cyclin expression has been implicated in oncogenesis in a number of human cancers. Since alterehd function of regulators of cyclin-dependent kinase (CDK) activity other than cyclins, in particular CDK inhibitors, might play a similar role in oncogenesis, we examined the expression and regulation of the CDK inhibitors p16^INk4 p15^INK4B and p21^WAF1/CIP1 in human breast cancer cell lines. Both the INK4 and INK4B genes were homozygously deleted in 3 cell lines, while INK4 alone was deleted in 2 cell lines. A further 2 cell lines displayed loss of an allele at this locus, and in 1 of these the remaining allele contained a mis-sense mutation within the coding region of the p16^INK4 protein. The majority of cell lines examined, including 2 normal mammary epithelial cell strains, expressed low levels of INK4 mRNA and low or undetectable levels of INK4B mRNA. However, INK4 mRNA was expressed at high levels in 5 cell lines, and this was associated with deletion or inactivation of the retinoblastoma susceptibility gene product pRB but not with mutation of TP53. No deletions of the WAF1/CIP1 gene were observed, but WAF1/CIP1 mRNA levels were reduced in cell lines with TP53 mutation. Transfection of a p16^INK4 expression vector into MDA-MB-231 cells lacking the INK4 gene failed to produce any p16^INK4-expressing cell lines, suggesting that such cells were selected against in continuous culture. Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas. Thus, homozygous deletion of the INK4 gene appears to be a rare event in primary breast cancer.     

Homozygous deletions of p16INK4 occur frequently in bilharziasis-associated bladder cancer

We have studied p16^INK4 mutation (by PCR-SSCP) and deletion (by Southern blotting and/or multiplex PCR) in a series of 47 bilharziasis-associated tumors from Egypt and compared the results with those obtained on a series of 17 established bladder cell lines and non-bilharziasis-associated bladder cancers from the Netherlands. In the cell lines we found 9 homozygous deletions and 1 mutation (59% of p16^INK4 alterations in cell lines), whereas in cases from the Netherlands deletions were found in 4 of 22 samples. No mutations were detected in the 46 samples screened. Interestingly, in bilharziasis-associated bladder cancer, deletions were present in 23 samples and mutations in a further 2 cases (53% of p16^INK4 alteration in bilharziasis-associated bladder cancer). No correlation was found between p16^INK4 alteration and histopathological data. Likewise, the same frequency of alteration was found in tumors with different differentiation patterns (squamous, transitional or adenocarcinoma). Three conclusions can be drawn from our findings: (i) p16^INK4 alterations are more frequent in cell lines than in primary tumors; (ii) in primary bladder tumors (bilharziasis-associated or not), p16^INK4 deletions are much more frequent than p16^INK4 mutations; (iii) p16^INK4 alterations are more frequent in bilharziasis-associated bladder tumors than in other bladder tumors. This high frequency of deletion is not related to a specific histological type but to the specific etiology of these tumors. © 1996 Wiley-Liss, Inc.     

Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay

BACKGROUND.

The aim of this study was to examine p16^INK4a protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16^INK4a Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high‐grade lesions was assessed by using follow‐up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high‐risk HPV (hc₂) results.

METHODS.

Three hundred ninety‐eight residual ThinPrep samples were collected, and histological follow‐up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou‐stained slide, a second slide was processed in preparation for p16^INK4a immunostaining. High‐risk human papillomavirus testing (hc₂) was also performed.

RESULTS.

Of the 163 cytologically abnormal samples, 6‐month biopsy follow‐up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow‐up were positive for p16^INK4a, yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16^INK4a negative, yielding a diagnostic specificity of 62%. In comparison, the hc₂ test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow‐up, 25 of 26 slides were deemed to be positive for p16^INK4a, increasing the diagnostic sensitivity to 96%.

CONCLUSIONS.

The CINtec p16^INK4a Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16^INK4a protein overexpression. Diagnostic specificity of the CINtec p16^INK4a assay was significantly improved relative to hc₂. To increase p16^INK4a immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.     

p19INK4d mRNA and protein expression as new prognostic factors in ovarian cancer patients

p19^INK4d (CDKN2D) is a negative regulator of the cell cycle. Little is known of its role in cancer development and prognosis. We aimed to evaluate the clinical significance of p19^INK4d expression in ovarian carcinomas with respect to the TP53 accumulation status, as well as the frequency of CDKN2D mutations. p19^INK4d and TP53 expression was evaluated immunohistochemically in 445 ovarian carcinomas: 246 patients were treated with platinum–cyclophosphamide (PC/PAC), while 199 were treated with taxane–platinum agents (TP). CDKN2D gene expression (mRNA) was examined in 106 carcinomas, while CDKN2D mutations in 68 tumors. Uni- and multivariate statistical analyses (logistic regression and the Cox proportional hazards model) were performed for patient groups divided according to the chemotherapeutic regimen administered, and in subgroups with and without TP53 accumulation. High p19^INK4d expression increased the risk of death, but only in patients with the TP53-negative carcinomas (HR 1.61, P = 0.049 for PC/PAC-treated patients, HR 2.00, P = 0.015 for TP-treated patients). This result was confirmed by the mRNA analysis (HR 4.24, P = 0.001 for TP-treated group). High p19^INK4d protein expression associated with adverse clinicopathological factors. We found no alterations in the CDKN2D gene; the c.90C>G (p.R30R; rs1968445) polymorphism was detected in 10% of tumors. Our results suggest that p19^INK4d expression is a poor prognostic factor in ovarian cancer patients. Analyses of tumor groups according to the TP53 accumulation status facilitate the identification of cancer biomarkers.     

Telomere dysfunction and inactivation of the p16(INK4a)/Rb pathway in pyothorax-associated lymphoma

Previous studies have indicated that genome instability is involved in the lymphomagenesis of pyothorax-associated lymphoma (PAL), which develops in patients with a long-standing history of pyothorax. One of the well-known causes of genome instability is telomere dysfunction. In the present study, the condition of telomeres was analyzed in the cell lines and clinical samples from PAL. Telomere length (TL) in PAL cell lines was extremely short (<4.5 kbp). TL in tumor samples was broad in range, and shorter than that in the peripheral blood leukocytes from the matched patients. Three of five PAL cell lines showed frequent loss of telomere signals (telomere erosion); however, telomerase activity in PAL cell lines was similar to that in Burkitt lymphoma cell lines. Rb expression was detected in three PAL cell lines and four of 15 clinical samples, respectively. Rb protein expressed in three PAL cell lines was heavily phosphorylated, indicating that function of Rb protein was suppressed. p16(INK4a) expression was not detected in either cell lines or clinical samples. The promoter region in p16(INK4a) was heavily methylated in all cell lines as well as the clinical samples. Inactivation of the p16(INK4a)/Rb pathway may allow continuous cell division and critical telomere shortening, which induce genome instability, finally leading to malignant transformation. Taken together, telomere dysfunction and inactivation of the p16(INK4a)/Rb pathway might play a role for PAL development.     

Molecular analysis of the cyclin-dependent kinase inhibitor genes p15INK4b/MTS21, p16INK4/MTS1, p18 and pl9 in human cancer cell lines

Cyclin-dependent kinase-4 inhibitor genes (INK4) regulate the cell cycle and are candidate tumor-suppressor genes. To determine if alterations in the coding regions of the pl8 and pl9 genes, which are novel members of the INK4 family and if they correlate with the development of human cancer, 100 human cancer cell lines were analyzed. Two other INK4 gene family members, p15^INK4b/MTS2 and pl6^INK4/MTS1 genes were also analyzed. Homozygous deletions of the p15^INK4b/MTS2 gene were detected in 29 cancer cell lines. Thirty-five homozygous deletions and 7 intragenic mutations of the pl6^INK4/MTS1 gene were also detected in these cell lines. Neither homozygous deletions nor intragenic mutations of the p18 and p19 genes were found except in an ovarian cancer cell line, SKOV3, harboring a single base pair deletion in exon 1 of p19. In p16^INK4/MTS1 expression analysis, 5 cell lines with both authentic and alternative spliced pl6^INK4/MTS1 mRNA had no detectable pl6^INK4/MTS1 protein. These results suggest the hypotheses that either post-translational modification or enhanced degradation may be responsible for the lack of detection of the pl6^INK4/MTS1 protein. Using Western blot analysis, subsets of 26 human cancer cell lines were examined for pl8 expression and 39 cell lines for p19 expression. All of these cell lines expressed the pl8 or pl9 protein, with the exception of SKOV3, which did not express pl9. Therefore, the INK4 gene family may be divided into 2 groups. One group includes p15^INK4b/MTS2 and pl6^INK4/MTS1 in which genetic and epigenetic alterations might contribute to the development of human cancers. The other group includes p18 and p19, in which somatic mutations are uncommon in many types of human cancer, and their role in human carcinogenesis and cancer progression is uncertain. (This article is a US Government work and, as such, is in the public domain in the United States of America.) © 1996 Wiley-Liss, Inc.     

Establishment,characterization and drug sensitivity of four new human soft tissue sarcoma cell lines

Four new cell lines were established from patients with soft tissue sarcomas. Drug sensitivity as well as genotypic characterization, which may be related to drug sensitivity in these cell lines, was determined. Karyotype, H-ras, c-myc and mutant p53 gene expression, Rb, G_I- and S-phase cyclins, E2F and major cyclin/CDK inhibitors such as p16 and p21 and p-glycoprotein were analyzed using cytogenetic, Northern blot and immunological methods. Drug sensitivity was determined using growth inhibition tests. These cell lines differed in their morphology and growth rates, forming colonies in soft agar with a cloning efficiency of 4.3–13.4%, and 3 of the 4 cell lines grew in nude mice. Cytogenetic analysis of cell lines revealed highly aneuploid karyotypes. Deletion and/or translocation of chromosome 17 was seen in HS-16, HS-18 and HS-30 cells, and both copies of chromosome 13 were lost or re-arranged in the HS-18 cell line. Mutant p53 protein was present in all 4 cell lines. HS-18 cells showed no expression of the Rb protein and high levels of expression of E2F, cyclin A, cyclin E and CDK2. HS-16 expressed a higher level of cyclin D than the other 3 cell lines. p21^waf1 expression was seen in all cell lines, but p16^ink4 was expressed only in HS-30 and HS-42 cell lines. These cell lines were sensitive to taxol and relatively resistant to methotrexate, vinblastine and 5-fluorouracil when compared with the fibrosarcoma cell line HT-1080. These new cell lines should provide a useful model for the study of soft tissue sarcomas and for evaluating new drugs or treatments. © 1996 Wiley-Liss, Inc.     

Homozygous deletion of CDKN2A (p16INK4a/p14ARF) but not within 1p36 or at other tumor suppressor loci in neuroblastoma

Loss of heterozygosity of several specific genomic regions is frequently observed in neuroblastoma tumors and cell lines, but homozygous deletion (HD) is rare, and no neuroblastoma tumor suppressor gene (TSG) has yet been identified. We performed a systematic search for HD, indicative of a disrupted TSG, in a panel of 46 neuroblastoma cell lines. An initial search focused on a well-characterized consensus region of hemizygous deletion at 1p36.3, which occurs in 35% of primary neuroblastomas. Each cell line was screened with 162 1p36 markers, for a resolution of 13 kb within the consensus 1p36.3 deletion region and 350 kb throughout the remainder of 1p36. No HDs were detected. This approach was expanded to survey 21 known TSGs, specifically targeting intragenic regions frequently inactivated in other malignancies. HD was detected only at the CDKN2A (p16INK4a/p14ARF) gene at 9p21 and was observed in 4 of 46 cell lines. The observed region of HD included all exons of both CDKN2A and the closely linked CDKN2B (p15INK4b) gene for cell lines LA-N-6 and CHLA-174, all exons of CDKN2A but none of CDKN2B for CHLA-179, and only 104 bp within CDKN2A exon 2 for CHLA-101. All four deletions are predicted to inactivate the coding regions of both p16INK4a and p14ARF. HD was observed in corresponding primary tumor samples for CHLA-101 and CHLA-174 but was not present in constitutional samples. These results suggest that for neuroblastoma, large HDs do not occur within 1p36, most known TSGs are not homozygously deleted, and biallelic inactivation of CDKN2A may contribute to tumorigenicity in a subset of cases.     

p16INK4a loss and sensitivity in KSHV associated primary effusion lymphoma

The Kaposi's Sarcoma associated Herpes virus (KSHV) encodes two genes with the potential to affect the activity of the retinoblastoma protein (Rb). Open reading frame (orf) 72 encodes a D type cyclin (kcyc) that can elicit p16INK4a resistant cdk activity and orf73 encodes the latency associated nuclear antigen (LNA) that can bind Rb and neutralize E2F regulation. This indicates that, like papilloma and adenovirus associated malignancies, those associated with KSHV are defective with respect to their Rb pathway. To address this we investigated whether KSHV associated primary effusion lymphoma (PEL) derived cell lines are resistant to growth inhibition by p16INK4a. We provide evidence that ectopic expression of p16INK4a in these cells causes an Rb dependent G1 cell cycle block. Importantly, endogenous p16INK4a expression is not detected in six PEL derived cell lines and four primary PEL samples and examination of the p16INK4a locus shows deletion in two out of six and hypermethylation in four out of six PEL lines. Treatment of the latter with the demethylating agent 5'-aza-2' deoxycytidine leads to re-expression of p16INK4a protein. Taken together these results suggest that p16INK4a loss may be a cellular change frequently associated with PEL. They furthermore argue that despite the presence of KSHV DNA and expression of a latent gene program Rb function is intact in PEL.     

p16INK4a is superior to high‐risk human papillomavirus testing in cervical cytology for the prediction of underlying high‐grade dysplasia

BACKGROUND

The primary goal of this study was to compare the clinical performance of an optimized and rigorously controlled immunocytochemical (ICC) assay for p16^INK4a to high‐risk (HR) human papillomavirus (HPV) detection by polymerase chain reaction (PCR) as diagnostic adjuncts for cytology specimens from colposcopy patients.

METHODS:

The study included 403 cervical cytology specimens collected within 3 months of colposcopy. The colposcopic impression and cervical biopsy diagnosis served as the standards for correlation with cytological, p16^INK4a, and HPV data. p16^INK4a was evaluated using an immunoperoxidase‐based assay that was linear over 4 logs for the detection of HeLa‐spiked positive control cytology specimens, using a threshold for positive test results that was based on receiver operating characteristic curve analysis. HR‐HPV was detected by multiplex PCR using genotype‐specific primers.

RESULTS:

In all combined diagnostic categories (negative for intraepithelial lesion and malignancy, atypical glandular cells, atypical squamous cells of undetermined significance, atypical squamous cells cannot exclude high‐grade squamous intraepithelial lesion, low‐grade squamous intraepithelial lesion, and high‐grade squamous intraepithelial lesion), the p16^INK4a ICC and HR‐HPV assays, respectively, had sensitivity of 81.7% and 83.3% (P = .81) and specificity of 78.1% and 50.9% (P < .001) for the detection of underlying ≥grade 2 cervical intraepithelial neoplasia (CIN) lesions on biopsy. Furthermore, the positive predictive value of p16^INK4a ICC was greater than that of HR‐HPV for patients with biopsies ≥CIN‐2 (41.2% and 24.2%, respectively, P = .001).

CONCLUSIONS:

This p16^INK4a immunocytochemical assay has superior specificity but similar sensitivity to HR‐HPV testing to predict underlying high‐grade dysplastic lesions in patients who are referred for colposcopy. The determination of the overall performance characteristics of p16^INK4a immunocytochemistry, as an independent test or in combination with HPV testing in low‐risk screening populations, however, will require subsequent large‐scale prospective clinical trials. Cancer (Cancer Cytopathol) 2010. © 2010 American Cancer Society.     

Growth arrest and suppression of tumorigenicity of bladder carcinoma cell lines induced by the P16/CDKN2 (p16INK4A,MTS1) gene and other loci on human chromosome 9

Wild-type P16/CDKN2 (p16^INK4A, MTSI) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequences. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32–33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression. © 1996 Wiley-Liss, Inc.     

p16INK4, pRB,p53 and cyclin D1 expression and hypermethylation of CDKN2 gene in thymoma and thymic carcinoma

There have been few reports on genetic alterations in thymomas. To investigate the expression of p16^INK4A, RB, p53 and cyclin D1 in thymomas, we first examined 36 thymomas (non-invasive type, 16 cases; invasive type, 20 cases) and 3 thymic carcinomas, using immunohistochemistry. Abnormal expression of p16^INK4A, RB, p53 and cyclin D1 was observed in 18, 8, 10 and 7 cases, respectively. Only a subgroup of invasive thymomas and thymic carcinomas showed an inverse correlation between p16^INK4A and RB expression. Subsequently, we examined the 36 thymomas and 4 thymic carcinomas for mutations in p53 and CDKN2 genes, using PCR-SSCP and direct-sequencing analyses. No mutation of these genes was detected in the thymomas and thymic carcinomas examined. A polymorphism in the 3′ untranslated region of exon 3 of CDKN2 was detected in 5 cases of thymoma. We searched for hypermethylation in the promoter region of CDKN2, observing it in 4 thymomas and 1 thymic carcinoma. Our data suggest that, unlike other more common cancers, alteration of the p53 gene may not play a significant role in the tumorigenesis of thymoma. However, inactivation of p16^INK4A and RB may play a role in the progression of thymoma and thymic carcinoma. Int. J. Cancer 73:639–644, 1997. © 1997 Wiley-Liss, Inc.     

Ratio of Expression of p16INK4a to p14ARF Correlates with the Progression of Non-small Cell Lung Cancer

The CDKN2 gene is located on the short arm of chromosome 9p and encodes two unrelated proteins, p16^INK4a and p14^ARF, through the use of independent first exons and shared exons 2 and 3. p16^INK4a is a cyclin‐dependent kinase inhibitor, whereas p14^ARF regulates the cell cycle through a p53 and MDM2–dependent pathway. We have examined the expression of p16^INK4a and p14^ARF using competitive RT‐PCR in 60 non‐small cell lung cancers (NSCLCs) and matching normal lung tissues. The intensities of bands for p16^INK4a and p14^ARF were nearly equal or the intensity of the p16^INK4a band slightly exceeded that of p14^ARF in the normal lung tissues (n=60). In 38 tumors the intensity of the p16^INK4a band was similar to or slightly weaker than that of p14^ARF. In 6 tumors the intensity of the p16^INK4a band was weaker than that of p14^ARF. In 15 tumors the intensity of the p14^ARF band was very strong and the p16^INK4a band was barely visible. In only one tumor was the intensity of the p16^INK4a band very strong, while the band of p14^ARF was barely visible. The ratio of the intensity of p16^INK4a to p14^ARF had an interesting correlation with the tumor's clinicopathological characteristics. The p stage II‐IV tumors had significantly lower p16^INK4a to p14^ARF ratios than the p stage I tumors (P=0.036). The T2–4 tumors had significantly lower p16^INK4a to p14^ARF ratios than the T1 tumors (P=0.005). The N1–3 tumors had significantly lower p16^INK4a to p14^ARF ratios than the NO tumors (P=0.014). Our results suggest that the ratio of expression of p16^INK4a to p14^ARFtends to decrease during the progression of NSCLC.     

Inhibition of T-cell acute lymphoblastic leukemia proliferation in vivo by re-expression of the p16INK4a tumor suppressor gene

T-cell acute lymphoblastic leukemia (T-ALL) is characterized by the presence of differentiation-inhibited pro- and pre-T-cell blasts. The p16^INK4a tumor suppressor gene has been shown to be frequently deleted in human T-ALL cases. Deletion of p16^INK4a may be associated with poor prognosis and relapse of the disease. Radiation-induced murine T-ALL in C57B1/6 mice shares pathogenetic and molecular characteristics with the human disease. We used the murine disease as a model to study the status of the INK4/ARF gene locus and to examine the effect of p16^INK4a-re-expression in T-ALL cells on their leukemic potential in vivo. In 9 of 17 radiation-induced murine T-ALL cell lines, the p16^INK4a protein was not expressed as determined by immunoblotting. Southern blot analysis revealed homozygous deletions of the p16^INK4a gene locus in three of the nine lines, along with the genes encoding p15^INK4b and p19^ARF. Transduction of p16^INK4a-negative T-ALL lines with retrovirus encoding p16^INK4a significantly inhibited their in vitro proliferation by inducing G₁-arrest. Importantly, re-expression of p16^INK4a in p16^INK4a-negative T-ALL cells obliterated the induction of lethal disseminated leukemia in syngeneic mice. This is the first demonstration that re-establishment of p16^INK4a expression is critical for in vivo growth regulation of T-ALL cells.     

Inversely correlated expression of p16 and Rb protein in non-small cell lung cancers: An immunohistochemical study

Cdk4-mediated phosphorylation of Rb protein is inhibited by p16, a product of a possible tumor suppressor gene. We examined the expression of p16 and Rb protein by means of immunohistochemistry in 61 non-small cell lung cancers and have demonstrated an inverse relationship between the expression of p16 and Rb protein: 28/30 specimens that did not stain for p16 stained for Rb and 21/31 p16-positive specimens did not stain for Rb. Only 1 of the p16-negative specimens had a mutation of exon 2 of the CDKN2 gene. Our results indirectly support the theory that p16 expression is negatively regulated by the functional Rb protein. © 1996 Wiley-Liss, Inc.