p16 mutations/deletions are not frequent events in prostate cancer.

Cyclin-dependent kinase-4 inhibitor gene (p16INK4) has recently been mapped to chromosome 9p21. Homozygous deletions of this gene have been found at high frequency in cell lines derived from different types of tumours. These findings suggested therefore, that p16INK4 is a tumour-suppressor gene involved in a wide variety of human cancers. To investigate the frequency of p16INK mutations/deletions in prostate cancer, we screened 20 primary prostate tumours and four established cell lines by polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) analysis for exon 1 and exon 2. In contrast to most previous reports, no homozygous deletions were found in prostate cancer cell lines, but one cell line (DU145) has revealed to a mutation at codon 76. Only two SSCP shifts were detected in primary tumours: one of them corresponds to a mutation at codon 55 and the other one probably corresponds to a polymorphism. These data suggest that mutation of the p16INK4 gene is not a frequent genetic alteration implicated in prostate cancer development.     

Mechanisms of inactivation of p14ARF, p15INK4b, and p16INK4a genes in human esophageal squamous cell carcinoma.

The 9p21 gene cluster, harboring growth suppressive genes p14ARF, p15INK4b, and p16INK4a, is one of the major aberration hotspots in human cancers. It was shown that p14ARF and p16INK4a play active roles in the p53 and Rb tumor suppressive pathways, respectively, and p15INK4b is a mediator of the extracellular growth inhibition signals. To elucidate specific targets and aberrations affecting this subchromosomal region, we constructed a detailed alteration map of the 9p21 gene cluster by analyzing homozygous deletion, hypermethylation, and mutation of the p14ARF, p15INK4b, and p16INK4a genes individually in 40 esophageal squamous cell carcinomas (ESCCs) and compared the genetic alterations with mRNA expression in 18 of these samples. We detected aberrant promoter methylation of the p16INK4a gene in 16 (40%), of p14ARF in 6 (15%), and of p15INK4b in 5 (12.5%) tumor samples. Most p16INK4a methylations were exclusive, whereas all but one of the p14ARF/p15INK4b methylations were accompanied by concomitant p16INK4a methylation. We detected homozygous deletion of p16INK4a in 7 (17.5%), of p14ARF-E1beta in 13 (33%), and of p15INK4b in 16 (40%) tumor samples. Most deletions occurred exclusively on the E1beta-p15INK4b loci. Two samples contained p14ARF deletion but with p16INK4a and p15INK4b intact. No mutation was detected in the p14ARF and p16INK4a genes. Comparative RT-PCR showed good concordance between suppressed mRNA expression and genetic alteration for p15INK4b and p16INK4a genes in the 18 frozen samples, whereas 5 of the 13 cases with suppressed p14ARF mRNA expression contained no detectable E1beta alteration but aberrations in the p16INK4a locus. Our results show that in human ESCCs, p14ARF is a primary target of homozygous deletion along with p15INK4b, whereas p16INK4a is the hotspot of hypermethylation of the 9p21 gene cluster. The frequent inactivation of the p14ARF and p16INK4a genes may be an important mechanism for the dysfunction of both the Rb and p53 growth regulation pathways during ESCC development.     

Deletion in p16INK4a and loss of p16 expression in human skin primary and metastatic melanoma cells

p16INK4a gene mapped at chromosome 9p21 region encodes a tumor suppressor protein p16 which is frequently inactivated in human cancers, including skin melanoma. In order to clarify the importance of p16 alterations in melanoma, we examined the deletions of p16INK4a and expression of p16 protein in eight unselected primary and metastatic melanoma cell lines from human skin melanomas. Normal skin melanocytes were used as controls. Deletions of entire exons in the p16INK4a gene were detected by PCR technique and expression of the p16 protein was examined by Western blotting and immunocytochemistry. Results showed that the fragments from exons 2A, 2C and 3 in p16INK4a gene were totally deleted in the metastatic melanoma cell line, FM28.7 and the fragment from exon 3 was deleted in the metastatic melanoma cell line, FM55M2. P16 protein was strongly expressed in two of the primary melanomas cell lines (FM55P and RaH3). The p16 protein was weakly expressed in one of the metastatic melanoma cell lines (FM55M1) and negative in the other metastasis (FM55M2) as compared to their matched primary melanoma cells (FM55P). The p16 protein was strongly expressed in normal skin melanocytes. Immunocytochemistry showed that p16 protein was mainly localized in the nuclei of the melanoma cells and normal melanocytes, if it was expressed. Deletions of p16INK4a gene was uncommon and loss of p16 protein expression was common event in melanoma, especially in the later stages of melanoma.     

Molecular analysis of INK4 genes in breast carcinomas

Cell cycle regulators have recently been implicated in oncogenic transformation of cells, including the cyclins active in the G1 phase of the cell cycle and their respective cyclin-dependent kinases (CDK) whose activities are regulated by a set of inhibitors of CDK (CDKI). Since CDKIs can inhibit cell proliferation, they may have a role as tumor suppressor genes. To determine if alterations of CDKI genes may be involved in tumorigenesis of breast cancer, we examined the mutational status of p16(INK4A), p15(INK4B), p18(INK4C), p19(INK4D) CDKI genes in 36 primary breast carcinomas and 9 breast cancer cell lines using polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP), direct DNA sequencing, and Southern blot analysis. Furthermore, amplification of cyclin D1, D2, D3 genes were also examined in these samples. One mutation of p15(INK4B) gene occurred, resulting in change of aspartic acid to asparagine at codon 85. Since aspartic acid at this position is conserved between all four human and murine INK4 proteins, this missense mutation may have functional significance. The sample with a p15(INK4B) point mutation was accompanied by amplification of the cyclin D1 gene. A deletion of the p18(INK4C) gene was found in a primary tumor. Three deletions of the p16(INK4A) gene and two deletions of the p15(INK4B) gene were found in the cell lines. Also, we found amplification of the p15(INK4B) and p16(INK4A) loci in a clinical sample as well as amplification of the p19(INK4D) in another sample, and amplification of the myeloperoxidase (MPO) gene in one cell line and two primary tumors. We suspect that a critical gene for breast cancer is amplified near the MPO gene. These data indicate that CDKI mutations are moderately rare in breast cancer and are often associated with the simultaneous alteration of more than one cell-cycle regulatory gene.     

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.     

Inactivation of the INK4a/ARF locus and p53 in sporadic extrahepatic bile duct cancers and bile tract cancer cell lines.

The tumor-suppressor genes p14(ARF), p16(INK4a) and Tp53 are commonly inactivated in many tumors. We investigated their role in the pathogenesis of 9 bile tract cancer cell lines and 21 primary sporadic extrahepatic bile duct carcinomas. p53 and p16 protein expression was examined by Western blot analysis and immunohistochemistry. Mutation screening of p53 was done by SSCP and direct sequencing. Inactivating mechanisms of p14 and p16 were addressed by screening for mutations, homozygous deletions, chromosomal loss of 9p21 (loss of heterozygosity [LOH] analysis) and promoter hypermethylation of the p14/p16 genes. p53 overexpression could be detected in 7 of 9 cell lines and 7 of 21 primary tumors, but mutations were found in 3 cell lines only. p16 expression was absent in all cell lines, due to homozygous deletion of the gene in 8 of 9 cell lines and hypermethylation of the p16 promoter in one cell line (CC-LP-1). p14 exon 1beta was homozygously deleted in 6 of 9 cell lines, while retained in CC-LP-1 and 2 additional lines. No p14 promoter hypermethylation could be detected. p16 expression was lost in 11 of 21 primary tumors. p16 promoter hypermethylation was present in 9 of 21 primary tumors, all with lost p16 expression. Allelic loss at 9p21 was detected in 13 of 21 primary tumors, 10 of 11 with lost p16 expression and 8 of 9 with methylated p16 promoter. No p14 promoter hypermethylation or p14/p16 mutations could be detected. Neither Tp53 nor p16 alterations showed obvious association with histopathologic or clinical characteristics. In conclusion, inactivation of the p16 gene is a frequent event in primary sporadic extrahepatic bile duct cancers, 9p21 LOH and promoter hypermethylation being the principal inactivating mechanisms. Therefore, p16, but not p14, seems to be the primary target of inactivation at the INK4a locus in bile duct cancers. Other mechanisms than Tp53 mutations seems to be predominantly responsible for stabilization of nuclear p53 protein in bile duct cancers.     

Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of p15(INK4B) tumor suppressor gene

A 42?kb region on human chromosome 9p21 encodes for three distinct tumor suppressors, p16(INK4A), p14(ARF) and p15(INK4B), and is altered in an estimated 30-40% of human tumors. The expression of the INK4A-ARF-INK4B gene cluster is silenced by polycomb during normal cell growth and is activated by oncogenic insults and during aging. How the polycomb is recruited to repress this gene cluster is unclear. Here, we show that expression of oncogenic Ras, which stimulates the expression of p15(INK4B) and p16(INK4A), but not p14(ARF), inhibits the expression of ANRIL (antisense non-coding RNA in the INK4 locus), a 3.8?kb-long non-coding RNA expressed in the opposite direction from INK4A-ARF-INK4B. We show that the p15(INK4B) locus is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated. Notably, depletion of ANRIL disrupts the SUZ12 binding to the p15(INK4B) locus, increases the expression of p15(INK4B), but not p16(INK4A) or p14(ARF), and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates that ANRIL binds to SUZ12 in vivo. Collectively, these results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15(INK4B) locus.     

Quantitative real-time PCR does not show selective targeting of p14(ARF) but concomitant inactivation of both p16(INK4A) and p14(ARF) in 105 human primary gliomas

In many human cancers, the INK4A locus is frequently mutated by homozygous deletions. By alternative splicing this locus encodes two non-related tumor suppressor genes, p16(INK4A) and p14(ARF) (p19(ARF) in mice), which regulate cell cycle and cell survival in the retinoblastoma protein (pRb) and p53 pathways, respectively. In mice, the role of p16(INK4A) as the critical tumor suppressor gene at the INK4A locus was challenged when it was found that p19(ARF) only knock-out mice developed tumors, including gliomas. We have analysed the genetic status of the INK4A locus in 105 primary gliomas using both microsatellite mapping (MSM) and quantitative real-time PCR (QRT-PCR). Comparison of the results of the two methods revealed agreement in 67% of the tumors examined. In discordant cases, fluorescence in situ hybridization (FISH) analysis was always found to support QRT-PCR classification. Direct assessment of p14(ARF) exon 1beta, p16(INK4A) exon 1alpha and exon 2 by QRT-PCR revealed 43 (41%) homozygous and eight (7%) hemizygous deletions at the INK4A locus. In 49 (47%) gliomas, both alleles were retained. In addition, QRT-PCR, but not MSM, detected hyperploidy in five (5%) tumors. Deletion of p14(ARF) was always associated with co-deletion of p16(INK4A) and increased in frequency upon progression from low to high grade gliomas. Shorter survival was associated with homozygous deletions of INK4A in the subgroup of glioblastoma patients older than 50 years of age (P=0.025, Anova test single factor, alpha=0.05).     

Increase in the frequency of p16INK4 gene inactivation by hypermethylation in lung cancer during the process of metastasis and its relation to the status of p53.

The p16INK4 gene, which is a tumor suppressor gene, is frequently altered in lung cancers. Hypermethylation of the promoter region of the p16INK4 gene seems to be the major mechanism through which p16INK4 become inactivated. Hypermethylation of the p16INK4 gene was reported to occur at an early stage in lung cancer. To determine whether the change in p16INK4 methylation status occurs at the late stage in the progression of primary lung cancers, we analyzed the primary and metastatic tumor tissues and normal lung samples from 29 cases of advanced lung cancer with distant metastasis. In each tissue sample, we analyzed the p16INK4 and p15INK4b genes for mutations and the methylation status of both genes using PCR-single strand conformation polymorphism, direct sequencing, and methylation-specific PCR analysis. We also analyzed a subset of the samples for p16INK4 protein expression. Genetic mutations in the coding region of the p16INK4 and p15INK4b genes were not found in any of the examined specimens. The promoter region of the p16INK4 gene was hypermethylated in the tumor samples of the primary or metastatic site of 37.0% (10 of 27) of the subjects. The promoter region of the p16INK4 gene was hypermethylated at both the primary and metastatic sites in two of the 10 cases and at only the metastatic site in 8 cases. By immunohistochemical analysis, we confirmed the presence of p16INK4 protein at the primary site of all cases in which the promoter region of the p16INK4 gene was hypermethylated at only the metastatic site. Interestingly, all 8 cases with a hypermethylated p16INK4 promoter region, at only the metastatic site, did not have p53 mutation. The results of this study indicate that tumor cells in which the p16INK4 gene has been inactivated by hypermethylation of the promoter region could have an advantage in progression and metastasis in non-small cell lung cancers, especially in the tumors with normal p53, and that the frequency of p16INK4 gene inactivation by hypermethylation could vary in clinical course.     

High frequency of homozygous deletion and methylation of p16(INK4A) gene in oral squamous cell carcinomas

p16(INK4A) inactivation was analyzed in ten squamous cell carcinoma (SCC) cell lines and 32 primary SCCs, using the polymerase chain reaction (PCR), PCR-single-strand conformation polymorphism, methylation-specific PCR, and cycle sequencing. In the study of cell lines, we detected three deletions in exon 1alpha and exon 2, and detected two methylations. Among tumor samples, we detected the homozygous deletions (HDs) of 43.8% in exon 1alpha 34.4% in exon 2, and methylation was found in 50.0%. The lack of p16(INK4A) with immunohistochemistry was detected in 71.9% and matched the alteration of p16(INK4A) gene. These results suggest that p16(INK4A) inactivation is predominantly caused by HD and methylation, and immunohistochemical evaluation of p16(INK4A) is a useful method.     

INK4a gene expression and methylation in primary breast cancer: overexpression of p16INK4a messenger RNA is a marker of poor prognosis.

Frequent deletions or mutations of the INK4 gene, which encodes the cyclin-dependent kinase 4 inhibitor p16INK4a, have been documented in various human cancers, but little is known about the role of this tumor suppressor gene in primary breast cancer. We examined p16INK4a mRNA expression and its relationship with cyclin D1 and estrogen receptor (ER) expression in 314 primary breast cancers using Northern blots probed with a p16 exon 1alpha-specific cDNA. Tumor samples overexpressing p16INK4a were predominantly ER negative with low levels of cyclin D1. Cyclin D1 and ER mRNA levels in the high p16INK4a expressers were significantly lower than those in the remainder of the population (P = 0.0001). Furthermore, the mean p16INK4a mRNA level in the ER-negative tumors was significantly higher than that in the ER-positive group (P = 0.0001). Because the INK4 gene is frequently inactivated by de novo methylation, we investigated the frequency of INK4a exon 1alpha methylation in a subset of 120 primary breast cancers using methylation-specific PCR; 24 of these were methylated. These findings indicate that high expression of p16INK4a and reduced expression due to de novo INK4a methylation are frequent events in primary breast cancer. In a subset of 217 patients for whom detailed clinical data were available, high p16INK4a mRNA expression was associated with high tumor grade (P = 0.006), > or = 4 axillary lymph node involvement (P = 0.004), ER negativity (P = 0.0001), and increased risk of relapse (P = 0.006). The significant negative correlation between p16INK4a and ER gene expression raises issues regarding their functional interrelationships and whether high p16INK4a expression may be associated with a lack of hormone responsiveness in 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.     

Chromosomal and genetic alterations of 7,12- Dimethylbenz[a]anthracene–induced melanoma from TP-ras transgenic mice

The TP-ras transgenic mouse line expresses an activated human T24 Ha-ras gene with a mutation in codon 12, regulated by a mouse tyrosinase promoter. The transgene is expressed in melanocytes of the skin, eyes, and brain. The mice develop cutaneous melanoma when treated with 7,12-dimethylbenz[a]anthracene. Cell lines have been generated from the cutaneous tumors and metastatic lesions. By using fluorescence in situ hybridization with mouse whole chromosome paints, the cell lines were characterized for chromosomal abnormalities. Key findings in the tumor cells included translocations of chromosome 4 and alterations in chromosome 6. One tumor cell line contained a double translocation involving chromosomes 3 and 6. To extend the results of the chromosome 4 painting, Southern analysis of the p15^INK4B, p16^INK4A, and p19^INK4D genes was performed. Our data indicated that there were homozygous and partial allelic deletions and polymorphisms in the region of chromosome 4 containing these genes, resulting in the absence or reduced expression of the p16 product. These findings are similar to those reported for human melanoma, and the TP-ras transgenic mouse may therefore be a valuable model for studying novel strategies for melanoma prevention and treatment. Mol. Carcinog. 20:78–87, 1997. © 1997 Wiley-Liss, Inc.     

Growth inhibition and induction of apoptosis by flavopiridol in rat lung adenocarcinoma, osteosarcoma and malignant fibrous histiocytoma cell lines

Flavopiridol is the potent inhibitor of cdks sharing its function with endogenous cdk inhibitors, and causes arrest at both the G1 and G2 phases of the cell cycle resulting in apoptosis in various tumor cell lines. Cyclin-dependent kinase inhibitor p16INK4a induces cell cycle arrest in G1 or G2 or both, and is inactivated in many malignant tumors. In this study, we focused on the effects of flavopiridol on chemically-induced rat lung adenocarcinoma, osteosarcoma and malignant fibrous histiocytoma (MFH) cell lines showing different pattern of p16INK4a status. The data demonstrated that flavopiridol inhibited cellular growth in a dose- and time-dependent manner, inducing apoptosis within 24 h in all cell lines at a concentration of 300 nM. The growth inhibition rate was the greatest for lung adenocarcinoma cells, lacking p16INK4a expression associated with methylation-mediated gene silencing; 83% at a concentration of 300 nM for 72-h treatment; while the growth of osteosarcoma and MFH cells, both expressing p16INK4a, were inhibited at similar levels; 54-61% for osteosarcoma and 61-64% for MFH cell lines. Then, we further investigated the influence of p16INK4a induction upon the effect of flavopiridol in p16INK4a-deficient lung adenocarcinoma cells. 5-aza 2'-deoxycytidine (5-Aza-CdR) induced p16INK4a expression and inhibited cellular growth in lung adenocarcinoma at a similar level to that with flavopiridol treatment. After the induction of p16INK4a expression by 5-Aza-CdR, the growth inhibition rates of flavopiridol in the p16INK4a-induced lung adenocarcinoma cells could not achieve comparable inhibition to that in the p16INK4a-deficient cells; the efficacy was reduced compared to original p16INK4a-deficient cells at each concentration of 50, 100 and 500 nM for 72-h treatment. These data indicate that flavopiridol shows cell type specific inhibition and possibly acts in a more compensatory manner for endogenous p16INK4a function in tumor cells having the aberrations of p16INK4a gene.     

Inactivation of p16INK4a expression in malignant mesothelioma by methylation

The molecular mechanisms of oncogenesis in mesothelioma involve the loss of negative regulators of cell growth including p16(INK4a). Absence of expression of the p16(INK4a) gene product is exhibited in virtually all mesothelioma tumors and cell lines examined to date. Loss of p16(INK4a) expression has also been frequently observed in more common neoplasms such as lung cancer as well. In a wide variety of these malignancies, including lung cancer, p16(INK4a) expression is known to be inactivated by hypermethylation of the first exon. In a survey of ten mesothelioma cell lines, one cell line (NCI-H2596) was identified as possessing loss of p16(INK4a) gene product following gene methylation. This methylation in these mesothelioma cells could be reversed, resulting in re-expression of p16(INK4a) protein, following the treatment of the cells with cytidine analogs, which are known inhibitors of DNA methylation. In previous clinical trials in mesothelioma, the cytidine analog dihydro-5-azacytidine (DHAC) has been found to induce clinical responses in approximately 17% of patients with mesothelioma treated with this drug, including prolonged complete responses. In addition, we identified evidence for methylation of p16(INK4a) in three of 11 resected mesothelioma tumor samples. When both cell lines and tumors are combined, inactivation of p16(INK4a) gene product expression following DNA hypermethylation was found in four of 21 samples (19%). We are further exploring the clinical significance of inhibition of methylation in mesothelioma by cytidine analogs. This may provide a potential treatment target in some mesothelioma tumors by inhibition of methylation.