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.     

Establishment of a novel human B-cell line (OZ) with t(14;18)(q32;q21) and aberrant p53 expression was associated with the homozygous deletions of p15INK4B and p16INK4A genes

The novel human pre-B cell line OZ was established from a patient with an aggressive form of non-Hodgkin's lymphoma. Karyotypic analysis of both the primary tumour and OZ cells revealed several marker chromosomes, including the t(14;18)(q32;q21) translocation, which involves the Bcl-2 gene, and alterations on chromosome 17p. Southern blot analysis found identical rearrangements in the 5′ region of Bcl-2 gene in the primary tumour and OZ cells. Homozygous deletions of the p15^INK4B and p16^INK4A genes, however, were present only in OZ cells. Western blot analysis detected aberrant small molecular-weight p53 proteins in both cell types. In addition, OZ cells no longer expressed the CD20 antigen. These findings suggest that Bcl-2 gene rearrangement and aberrant p53 expression resulted in the original B-cell tumour. A subsequent transforming event involving the p15^INK4B and p16^INK4A genes may have generated more immature cells with a growth advantage during in vitro culture. The genetic alterations involving p53, p15and p16^INK4A may be implicated in the aggressive form of t(14;18)(q32;q21)-bearing tumours and their poor prognosis. © 1997 John Wiley & Sons, Ltd.     

CDKN2 in HPV-positive and HPV-negative cervical-carcinoma cell lines

Human cervical cancers frequently contain retinoblastoma protein (Rb) that is inactivated by binding with human papilloma virus (HPV) E7 protein or through mutation. The CDKN2 gene encodes p 16^INK4 which inhibits cdk4-cyclin D phosphorylation of Rb, preventing the G₁-S transition. To determine whether abnormalities of CDKN2 occur in cervical-cancer cells, 11 cervical cell lines, including 8 HPV-positive cell lines, 2 HPV-negative cell lines containing mutant Rb, and one tumorigenic cell line derived from normal cervical cells following transfection with HPV-16 and v-H-ras (CX16-2HR), were analyzed. No cell line had a homozygous deletion of exon 1 or 2 of CDKN2, and only one cell line, CX16-2HR, had an altered DNA sequence, which represents a common polymorphism at codon 148. To exclude the possibility of other subtle inactivating mutations, immunoblot analysis of protein lysates was performed using a polyclonal anti-p 16^INK4 rabbit anti-serum. Abundant levels of normal-sized pl6^INK4 were observed in all cell samples. Thus, no alterations of CDKN2 were detected in these cervical cell lines. These results confirm that mutational inactivation of p 16^INK4 is a rare event in tumor samples with compromised Rb activity.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Detection of RB, p16/CDKN2 and p15(INK4B) gene alterations with immunohistochemical studies in human prostate carcinomas

To examine the status of cell cycle-inhibitory genes in human prostate carcinoma, we investigated alterations of RE (retinoblastoma), p16/CDKN2 and p15(INK4B) genes in 32 adenocarcinomas with immunohistochemistry. PCR-single-strand conformation polymorphism (SSCP) was used to examine all 27 exons of the RE gene, exons 1 to 3 of the p16/CDKN2 gene and exons 1 and 2 of the p15(INK4B) gene for mutations. Loss of heterozygosity (LOH) for the RE gene was probed by restriction fragment length polymorphism (RFLP) analysis. In addition, coordinate samples were subjected to immunohistochemical studies for reactivity to RE and p16 protein. The RE gene alterations were detected in 5 of the 32 tumors (16%); of these, only one mutation, a missense substitution, occurred within an exon. The remaining four single base insertions or deletions were found within introns of the RE gene and no mutational event was detected in its promoter region. LOH involving intron 17 of RB was detected in three cases of 10 informative tumors (30%). Intragenic mutations were also present in 3 of the 32 tumors in the p16/CDKN2 gene. In contrast, no mutational events were found in the p15(INK4B) gene in the tumors. Only one tumor had both a p16/CDKN2 mutation and LOH of the RE gene. Expression of pRB was absent or reduced in 16 cancers, while p16 expression was present in all cases to varying degrees. The results suggest that p16/CDKN2 gene mutations occur rarely and intragenic mutation, but not LOH,of the RE gene is not required in prostatic tumorigenesis.     

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.     

Rare mutations and no hypermethylation at the CDKN2A locus in epithelial ovarian tumours

The tumour-suppressor gene CDKN2A (p16, MTS1, CDK4I) encodes a cell cycle-regulatory protein and is located on chromosome 9p21, a region deleted in a wide variety of human cancers. To determine the role of the CDKN2A gene in the development of ovarian adenocarcinomas, we examined a large series of benign, low malignant potential (LMP) and invasive ovarian neoplasms for evidence of loss of heterozygosity (LOH), homozygous deletions, point mutations and hypermethylation of the CDKN2A locus. We have previously reported LOH on 9p in 45% of malignant ovarian neoplasms and a smaller percentage of benign and LMP tumours. In the current study, 6 malignant tumours were identified with partial deletions of 9p21. In 5 of these, the CDKN2A gene lays within the minimal deleted region. Homozygous deletions of CDKN2A were observed in only 2/88 invasive ovarian tumours and in 5/11 ovarian cancer cell lines. Of 15 primary ovarian tumours analyzed, one nonsense mutation was identified in a mucinous LMP tumour. No evidence of hypermethylation of the CDKN2A gene was found in 50 primary ovarian adenocarcinomas nor in 3 ovarian cancer cell lines. In conclusion, homozygous deletions, mutations and the de novo methylation of 5′ CpG island are not frequent modes of inactivation of the CDKN2A gene in ovarian cancer. The target of 9p LOH in ovarian adenocarcinomas is therefore unknown. Int. J. Cancer 70:508–511. © 1997 Wiley-Liss Inc.     

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.     

A Comparative Study of Glioma Cell Lines for p16, p15, p53 and p21 Gene Alterations

A total of 10 glioma cell lines were examined for alterations of the p16, p15, p53 and p21 genes, which are tumor suppressor genes or candidates with direct or indirect CDK-inhibitory functions. Genetic alterations (deletions or mutations) were frequently seen in the p16, p15 and p53 genes in these cell lines, but not in the p21 gene. When the states of the p16, p15 and p53 genes were compared among cell lines, all the cell lines showed abnormalities in at least 1 gene, often in 2 or 3 genes coincidentally, suggesting that dysfunction of these genes is closely related to glioma cell growth. Although alteration of all 3 genes was most frequent, there were cell lines having either p16/p15 or p53 or p16 and p53 gene alterations, suggesting that the time order of these genetic alterations was variable depending on the cell line. Among cell lines examined, one with homozygous p53 gene deletion seemed of particular practical value, since such a cell line might be useful in various studies, including investigation of the functions of various mutant p53 genes in the absence of heteromeric protein formation. On examination of the primary tumor tissues, the same alterations of the p16/p15 and p53 genes as detected in the cell lines were demonstrated in all 6 cases examined: p16/p15 gene deletion in 1, p16 gene mutation in 1 and p53 gene mutations in 5 cases. This suggested that the p16/p15 and the p53 gene alterations and their combinations in at least some glioma cell lines reflected those in the primary glioma tissues.     

Absence of p51 alteration in human ovarian cancer

The p51 gene encodes a protein with significant homology to p53, thus, it is a candidate tumor suppressor gene. To investigate the involvement of the p51 gene in human ovarian carcinogenesis, p51 gene alterations were examined in primary ovarian cancers and ovarian cancer cell lines. Mutation analysis of the p51 gene was performed in 40 primary ovarian cancers and 6 ovarian cancer cell lines using the PCR-SSCP and direct sequencing methods. Expression of p51 mRNA was examined in 9 primary ovarian cancers and 5 ovarian cancer cell lines by Northern blot and RT-PCR analyses. No mutations of the p51 gene causing amino acid substitutions or frameshifts were detected in either primary tumors or cancer cell lines by PCR-SSCP analysis of the entire coding region, although several genetic polymorphisms were detected in three samples. Allelic imbalance was detected in 3 of 19 (16%) primary ovarian cancers. No p51 gene expression was detected in 9 primary ovarian cancers and the corresponding normal ovarian tissues by Northern blot and by RT-PCR analyses. One of 5 ovarian cancer cell lines showed p51 gene expression by Northern blot analysis (20%). These results indicated that p51 gene expression was silent in normal ovarian tissues and primary ovarian cancers, and that mutation of the p51 gene does not play a major role in the development of ovarian cancer.     

Cancer-associated mis-sense and deletion mutations impair p16INK4 CDK inhibitory activity

The p16^INK4 gene is a candidate tumour-suppressor gene which maps to the genomic locus 9p21, and mutations of this gene are associated with melanoma and other cancers. Biochemical studies suggest that p16^INK4 mediates its effects by specifically inhibiting the G₁ cyclin-dependent kinases CDK4 and CDK6, thereby regulating progression through G₁ into S phase of the cell cycle. To evaluate the functional effects of mutations in p16^INK4 which have been observed in primary cancers and cancer cell lines, we constructed a series of deletion mutants comprising amino acid regions 9–72, 9–131, 73–131 and 73–156; a mis-sense mutation identified in melanoma (ArgB7Pro); and the polymorphism Ala148Thr and investigated their ability to inhibit cyclin D1/CDK4 kinase activity in vitro. Removal of 25 amino acids from the carboxy terminus of p16^INK4 (9–131) had little impact on its inhibitory activity. In contrast, deletion of the 65 N-terminal amino acids comprising the first and second ankyrin repeats of p16^INK4 (73–131) abolished its inhibitory activity. The carboxy (73–156) and amino terminal (9–72) fragments of p16^INK4 also failed to inhibit cyclin D1/CDK4 activity. These results indicate that the core region (73–131) as well as amino acids N-terminal of this sequence are important, whereas sequences C-terminal of amino acid 131 are less important for the inhibitory activity of this molecule. The melanoma-associated Arg87Pro mutation resulted in loss of inhibitory activity, whereas the Ala148Thr polymorphic variant was as effective as the alanine variant of p16^INK4 in inhibiting D1/CDK4 kinase activity. Binding assays revealed that inhibition was invariably associated with p16^INK4 binding to CDK4. Hence, our studies indicate that minor perturbations in p16^INK4 primary structure can lead to loss of its inhibitory activity, possibly contributing to oncogenesis in numerous cell types. © 1996 Wiley-Liss, Inc.