Mutational analysis of Chk1, Chk2, Apaf1 and Rb1 in human malignant melanoma cell lines

Four tumour suppressor genes (Chk1, Chk2, Apaf1 and Rb1) in nine human malignant melanoma cell lines were screened for a loss of gene expression, point mutations and small deletions/insertions by cDNA-based DGGE/SCCP analysis. In two cell lines alterations of the investigated genes could be demonstrated. This result confirms our assumption of the participation of dysfunctional p53 inducer/effector genes in human melanoma aetiology. Furthermore, it points towards the probable principal role of diverse alternative p53-pathway disruption mechanisms in this highly therapy-resistant malignancy without affecting p53 itself. To our knowledge, this is the first CHK1/CHK2 mutation screening in human melanoma.     

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.     

Clinico-pathological features and somatic gene alterations in refractory ceramic fibre-induced murine mesothelioma reveal mineral fibre-induced mesothelioma identities

Although human malignant mesothelioma (HMM) is mainly caused by asbestos exposure, refractory ceramic fibres (RCFs) have been classified as possibly carcinogenic to humans on the basis of their biological effects in rodents' lung and pleura and in cultured cells. Hence, further investigations are needed to clarify the mechanism of fibre-induced carcinogenicity and to prevent use of harmful particles. In a previous study, mesotheliomas were found in hemizygous Nf2 (Nf2(+/-)) mice exposed to asbestos fibres, and showed similar alterations in genes at the Ink4 locus and in Trp53 as described in HMM. Here we found that Nf2(+/-) mice developed mesotheliomas after intra-peritoneal inoculation of a RCF sample (RCF1). Clinical features in exposed mice were similar to those observed in HMM, showing association between ascite and mesothelioma. Early passages of 12 mesothelioma cell cultures from ascites developed in RCF1-exposed Nf2(+/-) mice demonstrated frequent inactivation by deletion of genes at the Ink4 locus, and low rate of Trp53 point and insertion mutations. Nf2 gene was inactivated in all cultures. In most cases, co-inactivation of genes at the Ink4 locus and Nf2 was found and, at a lower rate, of Trp53 and Nf2. These results are the first to identify mutations in RCF-induced mesothelioma. They suggest that nf2 mutation is complementary of p15(Ink4b), p16(Ink4a) and p19(Arf) or p53 mutations and show similar profile of gene alterations resulting from exposure to ceramic or asbestos fibres in Nf2(+/-) mice, also consistent with the one found in HMM. These somatic genetic changes define different pathways of mesothelial cell transformation.     

Alterations of the p16(INK4) locus in human malignant mesothelial tumors

The INK4 locus has two promoters and encodes two unique proteins that share exons in different reading frames, p16(INK4a) and p14(ARF). The p16(INK4a) protein, by inhibiting cyclin-dependent kinase, down regulates Rb-E2F and leads to cell cycle arrest in the G1 phase. The p14(ARF) protein interacts with the MDM2 protein, neutralizing MDM2-mediated degradation of p53. Since p53/Rb genes are not altered in malignant mesothelioma, additional components of these pathways, such as p16(INK4a) and p14(ARF), are candidates for inactivation. In this study, we have examined p16(INK4a) and p14(ARF) alterations (gene deletion, mutation and promoter methylation) in 45 primary malignant mesothelioma specimens. Fourteen patients (31%) had altered p16; four tumors had a methylated promoter region (8.8%), 10 tumors showed p16 to be deleted (22.2%), and one tumor had a point mutation (2%). We did not find any instances of methylation in the p14(ARF) 5'-CpG island. Patients whose tumors had p16 deletion were significantly younger than those with methylation, and, in the patients whose lungs were studied for the prevalence of asbestos fibers, those with any p16 alteration had lower fiber counts than those with no p16 alteration. Hence, p16 gene alteration is relatively common in malignant mesothelioma, while p14(ARF) is rarely, if ever, methylated. Our data suggest that deletion of p16 occurs in a relatively susceptible subset of the population.     

High levels of MDM2 are not correlated with the presence of wild-type p53 in human malignant mesothelioma cell lines.

Prior analysis of 20 human mesothelioma cell lines for p53 status revealed only two mutations and one p53 null cell line, although p53 expression was detected in most cell lines. In addition, mRNA and protein expression of the retinoblastoma gene product in human mesothelioma cell lines is similar to normal controls. We have tested for p53 induction after exposure to ionising radiation and demonstrate this induction and, to a lesser extent, p21(WAF1) induction, in both normal mesothelial cells and p53-positive mesothelioma cell lines. We postulated that high levels of MDM2 might alter p53 and retinoblastoma tumour-suppressor function in mesothelioma. However, Southern blot analysis for mdm2 indicated that no amplification had occurred in 18 mesothelioma cell lines tested. Steady-state mRNA and protein levels also did not indicate overexpression. These results indicate that high levels of MDM2 are not responsible for inactivating the functions of wild-type p53 or the retinoblastoma gene product during the pathogenesis of malignant mesothelioma.     

p14(ARF) modulates the cytolytic effect of ONYX-015 in mesothelioma cells with wild-type p53

ONYX-015 has been reported to kill selectively tumor cells lacking functional p53. Genetic alterations of INK4a/ARF locus, which is a predominant event in malignant pleural mesothelioma, may result in loss of p14(ARF) and subsequent disruption of p53 pathway in cancer cells. In the present study, ONYX-015 was able to kill three mesothelioma cell lines (H28, H513, and 211H) with wild-type p53 but lacking p14(ARF). In contrast, MS-1 mesothelioma cells, which expressed both p53 and p14(ARF), were resistant to ONYX-015. Introducing p14(ARF) gene into the H28 cell, a mesothelioma cell without p14(ARF) expression, significantly increased the resistance of this cell line to the cytolytic effect of ONYX-015. Our results suggest that human mesotheliomas with wild-type p53 yet lacking p14(ARF) are potential candidates for ONYX-015 therapy.     

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.     

Gene therapy of established mesothelioma xenografts with recombinant p16INK4a adenovirus

The absence of expression of the p16INK4a gene product is observed in virtually all mesothelioma tumors and cell lines, whereas wild-type pRB expression is maintained. We have examined the potential therapeutic role of re-expressing the p16INK4a gene product in mice with established human mesothelioma xenografts. Experiments using Adp16 treatments in mesothelioma xenografts demonstrated prolonged survival and potential cure following treatment with p16INK4a-based gene therapy. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.     

Molecular analysis of tumor suppressor genes, Rb, p53, p16INK4A, p15INK4B and p14ARF in natural killer cell neoplasms.

Natural killer (NK) cell neoplasms, which are derived from mature or precursor NK cells, are rare diseases and are observed predominantly in Asian countries. We analyzed the status of the Rb, p53, p15INK4B, p16INK4A and p14ARF genes in these diseases by Southern blot, polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and western blot analysis. We used 31 NK cell neoplasms, including four cell lines derived from NK cell neoplasms, 3 myeloid / NK cell precursor acute leukemias, 4 blastic NK cell lymphoma / leukemias, 4 aggressive NK cell leukemia / lymphomas, 4 nasal NK cell lymphomas, and 12 chronic NK lymphocytosis. We found gene amplification of the p53 gene in one nasal NK cell lymphoma, and point mutations of the p53 gene in one blastic NK cell lymphoma / leukemia and one chronic NK lymphocytosis. In addition, homozygous deletions of p15, p16 and p14 genes in 5 out of 31 samples were detected; 3 were from nasal NK cell lymphoma and 2 from blastic NK cell lymphoma / leukemia. Also hemizygous deletion of the Rb gene in one blastic NK cell lymphoma was detected. Rb proteins were highly expressed in one cell line as well as two myeloid / NK cell precursor acute leukemias. In other cell lines, complete loss and an aberrant migration pattern of Rb protein expression were observed. Comparative genomic hybridization suggested that the homozygous deletions of the p15, p16 and p14 were subtle chromosomal deletions and could not be identified by standard karyotyping in some cases. Although the number of cases we analyzed was not large, alterations identified in the Rb, p53, p16, p15 and p14 genes are of significance and might be associated with tumorigenesis in NK cell neoplasms.     

Upregulated p53 expression activates apoptotic pathways in wild-type p53-bearing mesothelioma and enhances cytotoxicity of cisplatin and pemetrexed

The majority of malignant mesothelioma possesses the wild-type p53 gene with a homologous deletion of the INK4A/ARF locus containing the p14(ARF) and the p16(INK4A) genes. We examined whether forced expression of p53 inhibited growth of mesothelioma cells and produced anti-tumor effects by a combination of cisplatin (CDDP) or pemetrexed (PEM), the first-line drugs for mesothelioma treatments. Transduction of mesothelioma cells with adenoviruses bearing the p53 gene (Ad-p53) induced phosphorylation of p53, upregulated Mdm2 and p21 expression levels and decreased phosphorylation of pRb. The transduction generated cleavage of caspase-8 and -3, but not caspase-9. Cell cycle analysis showed increased G0/G1- or G2/M-phase populations and subsequently sub-G1 fractions, depending on cell types and Ad-p53 doses. Transduction with Ad-p53 suppressed viability of mesothelioma cells and augmented the growth inhibition by CDDP or PEM mostly in a synergistic manner. Intrapleural injection of Ad-p53 and systemic administration of CDDP produced anti-tumor effects in an orthotopic animal model. These data collectively suggest that Ad-p53 is a possible agent for mesothelioma in combination with the first-line chemotherapeutics.     

Adenovirus-mediated p14(ARF) gene transfer in human mesothelioma cells

BACKGROUND: The p14(ARF) protein encoded by the INK4a/ARF locus promotes degradation of the MDM2 protein and thus prevents the MDM2-mediated inhibition of p53. Homozygous deletion of the INK4a/ARF locus is common in human mesothelioma and may result in the loss of p14(ARF) and the inactivation of p53. We designed this study to evaluate the biologic and potential therapeutic roles of p14(ARF) expression in mesothelioma cells. Methods and Results: We constructed Adp14, an adenoviral vector carrying human p14(ARF) complementary DNA, and used it to transfect human mesothelioma cell lines H28, H513, H2052, and MSTO-211H. Overexpression of p14(ARF) led to increased amounts of p53 and the p21(WAF) proteins and dephosphorylation of the retinoblastoma protein. The growth rate of mesothelioma cells was inhibited markedly by infection with Adp14 compared with mock infection or infection with a control adenovirus vector, AdCtrl. Overexpression of p14(ARF) induced G(1)-phase cell cycle arrest and apoptotic cell death. Cytotoxicity assays showed that Adp14 had a statistically significantly (P =.002) greater effect on colon cancer (HCT116) cell lines containing two copies of the wild-type p53 gene than on p53-null cells, suggesting that functional p53 is a critical determinant of p14(ARF)-mediated cytotoxicity. CONCLUSIONS: The transfection of p14(ARF) into mesothelioma cells led to the overexpression of p14(ARF), which resulted in G(1)-phase arrest and apoptotic cell death. These results suggest that this gene therapy-based approach may be of use in the treatment of mesothelioma.     

Quinidine impairs proliferation of neurofibromatosis type 2-deficient human malignant mesothelioma cells

BACKGROUND: Human malignant mesotheliomas (HMMs) are aggressive tumors that arise from the mesothelium. They respond poorly to conventional tumor treatment and outcome is often fatal. Inactivating mutations of the neurofibromatosis type 2 (NF2) tumor suppressor gene merlin have been described in nearly 60% of primary malignant mesothelioma and in approximately 20% of the mesothelioma cell lines. Studies regarding human NF2 schwannoma cells revealed a higher proliferation and a larger noninactivating K(+) outward current compared with controls. The enhanced proliferation of merlin-deficient NF2 schwannoma cells could be reduced in the presence of quinidine, a K(+) channel blocker, whereas the proliferation of normal Schwann cells is not affected. The current study was undertaken to evaluate the effect of quinidine on the proliferation of HMM cell lines in relation to their NF2 status. METHODS: Proliferation analyses using bromodeoxyuridine incorporation was performed by immunocytochemical staining and fluorescence assisted cell sorting. The patch-clamp technique was applied for electrophysiologic characterization of the HMM cell lines. The cytochrome P450 2D6 locus, known to be mutated at high frequencies in NF2 patients and to be specifically inhibited by quinidine, was screened for mutations by cycle sequencing. RESULTS: Quinidine selectively reduces the proliferation of merlin-deficient HMM cell lines by causing a G(0)/G(1) arrest, whereas the proliferation rates of merlin-expressing HMM cell lines remain unchanged. The effect of quinidine on the proliferation of HMM cell lines appears to be correlated with the NF2 gene status but not with the K(+) outward current. No relation to cytochrome P450 2D6 mutations was detected. CONCLUSIONS: Quinidine or quinidine analogs are of potential therapeutic interest for the subset of merlin-deficient mesothelioma tumors.     

Molecular Analysis of Tumor Suppressor Genes, Rb, p53,pl6INK4A, pl5INK4B and p14ARF in Natural Killer Cell Neoplasms

Natural killer (NK) cell neoplasms, which are derived from mature or precursor NK cells, are rare diseases and are observed predominantly in Asian countries. We analyzed the status of the Rb, p53, p15INK4B, p16INK4A and p14ARF genes in these diseases by Southern blot, polymerase chain reaction–single strand conformational polymorphism (PCR–SSCP) and western blot analysis. We used 31 NK cell neoplasms, including four cell lines derived from NK cell neoplasms, 3 myeloid/ NK cell precursor acute leukemias, 4 blastic NK cell lymphoma/leukemias, 4 aggressive NK cell leukemia/lymphomas, 4 nasal NK cell lymphomas, and 12 chronic NK lymphocytosis. We found gene amplification of the p53 gene in one nasal NK cell lymphoma, and point mutations of the p53 gene in one blastic NK cell lymphoma/leukemia and one chronic NK lymphocytosis. In addition, homozygous deletions of pl5,p16 and p14 genes in 5 out of 31 samples were detected; 3 were from nasal NK cell lymphoma and 2 from blastic NK cell lymphoma/leukemia. Also hemizygous deletion of the Kb gene in one blastic NK cell lymphoma was detected. Rb proteins were highly expressed in one cell line as well as two myeloid/NK cell precursor acute leukemias. In other cell lines, complete loss and an aberrant migration pattern of Rb protein expression were observed. Comparative genomic hybridization suggested that the homozygous deletions of the p15, p16 and p14 were subtle chromosomal deletions and could not be identified by standard karyotyping in some cases. Although the number of cases we analyzed was not large, alterations identified in the Rb, p53, p16, p15 and p14 genes are of significance and might be associated with tumorigenesis in NK cell neoplasms     

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.     

Alterations in the p16(INK4a) and p53 tumor suppressor genes of hTERT-immortalized human fibroblasts

Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events. One cell line also acquired mutations in both alleles of the p53 tumor suppressor gene. Downregulation of p16(INK4a) and loss of wild-type p53 expression occurred after escape from crisis, so these mutations are most likely not required for immortalization of these cells but rather were selected for during continuous growth in vitro. These findings emphasize the need for caution in the use of hTERT-immortalized cells in studies of normal cell biology or in tissue engineering and the need to monitor for genetic instability and the accumulation of mutations in both the p16(INK4a)/pRb and p53 pathways.     

Association of p16(INK4a) and pRb inactivation with immortalization of human cells

To examine the association of cell cycle regulatory gene inactivation with human cell immortalization, we determined the expression status of INK4a, Rb, and WAF1/ CIP1, in eleven in vitro immortalized human cell lines, including fibroblasts and keratinocytes. Two human papillomavirus type 16 E6 expressing cell lines with telomerase activity, including a fibroblast cell line and a keratinocyte cell line, expressed no detectable p16(INK4a). These cell lines had a hyperphosphorylated pRb and reduced expression of p21(WAF1/CIP1). All of seven fibroblast cell lines immortalized either spontaneously or by (60)Co, X-rays, 4-nitroquinoline 1-oxide or aflatoxin B(1), maintaining their telomeres by the ALT (alternative lengthening of telomeres) pathway, displayed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. Levels of p21(WAF1/CIP1) expression varied among the cell lines. Two fibroblast cell lines that became immortalized following infection with a retrovirus vector encoding human telomerase catalytic subunit (hTERT) cDNA were also accompanied by inactivation of p16(INK4a) and pRb pathways. Acquisition of telomerase activity alone was not sufficient for immortalization of these cell lines. Taken together, all the cell lines including fibroblasts and keratinocytes, with either telomerase activity or the ALT pathway for telomere maintenance showed loss of expression of p16(INK4a) and hyperphosphorylation of pRb. These demonstrate the association of inactivation of both p16(INK4a) and pRb with immortalization of human cells including fibroblasts and epithelial cells and telomerase-positive cells and ALT-positive cells.     

Adenovirus-mediated Ink4a/ARF gene transfer significantly suppressed the growth of pancreatic carcinoma cells

OBJECTIVES: To determine the effects of adenovirus-mediated transfer of p14(ARF) and p16(INK4a) on growth and apoptosis of human pancreatic carcinoma cell lines. RESULTs: Pancreatic carcinoma cell lines, PC-7, PANC-1 and MIA PaCa-2 (p14(ARF)-/- and p16(INK4a)-/-), were used. PC-7 (p53 wt) and MIA PaCa-2 (p53 mt) cells infected with recombinant adenovirus vector expressing p14(ARF) gene (Adp14) showed significant inhibition of cell growth compared with control vector in vitro and in vivo, whereas PANC-1 cells (p53 mt) did not show such effects. Those three cell lines exhibited growth retardation and senescence phenotype after infected with the adenovirus vector containing p16(INK4a) gene (Adp16) in vivo and in vitro. CONCLUSIONS: Adenovirus-mediated transfer of p14(ARF) and p16(INK4a) produces significant growth suppression of pancreatic carcinoma cells in vitro and in vivo. The effects of Adp14 partly depend on the status of p53 gene in those cells.     

Genetic status of cell cycle regulators in squamous cell carcinoma of the oesophagus: the CDKN2A (p16(INK4a) and p14(ARF) ) and p53 genes are major targets for inactivation

We determined inactivation of the CDKN2A (p16(INK4a) and p14(ARF)) gene in 21 cases of oesophageal squamous cell carcinoma (OSCC). The tumours were also analysed for mutations in exons 5-8 and allelic losses in the p53 gene. In addition, we screened the CDKN2B (p15 INK4b), CDKN2C (p18 INK4c), CDK4 and p53R2 genes for mutations in the tumour tissues. Besides concomitant alterations in the CDKN2A and p53 loci in more than half of the cases, our results showed that in 18 OSCC (86%) the CDKN2A (p16(INK4a) and p14(ARF) ) gene was affected through mutations, homozygous/hemizygous deletions and promoter hypermethylation. Eight out of 10 tumours with mutations or promoter hypermethylation specific to the CDKN2A/p16 INK4a gene showed loss of the wild-type allele. One tumour with a single base deletion in the N-terminus (codon 8) of the CDKN2A/p16(INK4a) gene carried a novel germ-line mutation or a rare polymorphism (Ile51Met) in exon 2 of the CDK4 gene. Promoter hypermethylation in the CDKN2A/p14 ARF gene was detected in 11 tumours. In the p53 gene 15 mutations were detected in 14 tumours. We detected an inverse relationship between CDKN2A/p16 INK4a inactivation and frequency of loss of heterozygosity at the p53 locus (OR 0.09, 95% CI 0.01-0.98; Fisher exact test, P-value approximately 0.03). Screening of nine exons of the p53R2 [Human Genome Organisation (HUGO) official name RRM2B] gene resulted in identification of a novel polymorphism in the 5' untranslated region, which was detected in four cases. Our results suggest that the CDKN2A (p16(INK4a) and p14(ARF) ) and p53 genes involved in the two cell cycle pathways are major and independent targets of inactivation in OSCC.     

Role of tumor suppressor genes in the development of adult T cell leukemia/lymphoma (ATLL).

Adult T cell leukemia/lymphoma (ATLL) is one of the peripheral T cell malignant neoplasms strongly associated with human T cell leukemia virus type-I (HTLV-I). Although the viral transactivating protein Tax has been proposed to play a critical role in leukemogeneis as shown by its transforming activity in various experimental systems, additional cellular events are required for the development of ATLL. One of the genetic events in ATLL is inactivation of tumor suppressor genes. Among many candidates for tumor suppressor genes, the main genetic events have been reported to center around the cyclin-dependent kinase inhibitors ((CDKIs) p15INK4A, p16INK4B, p18INK4C, p19INK4D, p21WAF1, p27KIP1, and p57KIP2), p53 and Rb genes; all of them play a major regulatory role during G1 to S transition in the cell cycle. Acute/lymphomatous ATLL has frequent alterations of p15 (20%) and p16 (28-67%), while chronic/smoldering ATLL has fewer abnormalities of p15 (0-13%) and p16 (5-26%). Most of these changes are deletion of the genes; fewer samples have mutations. ATLL patients with deleted p15 and/or p16 genes have significantly shorter survival than those individuals with both genes preserved. Although genetic alterations of p18, p19, p21, p27 have rarely been reported, inactivation of these genes may contribute to the development of ATLL because low expression levels of these genes seem to mark ATLL. The p53 gene is mutated in 10-50% of acute/lymphomatous ATLL. Functional impairment of the p53 protein, even if the gene has wild-type sequences, has been suggested in HTLV-I infected cells. Each of these genetic events are mainly found in acute/lymphomatous ATLL, suggesting that alterations of these genes may be associated with transformation to an aggressive phenotype. The Rb tumor suppressor gene is infrequently structurally altered, but one half of ATLL cases have lost expression of this key protein. Notably, alterations of one of the CDKIs, p53 and Rb genes appear to obviate the need for inactivation of other genes in the same pathway. A novel tumor suppressor gene on chromosome 6q may also have a critical role in the pathogenesis of ATLL. Taken together, tumor suppressor genes are frequently altered in acute/lymphomatous ATLL and their alteration is probably the driving force fueling the transition from chronic/smoldering to acute/lymphomatous ATLL.     

G1 checkpoint protein and p53 abnormalities occur in most invasive transitional cell carcinomas of the urinary bladder.

The G1 cell cycle checkpoint regulates entry into S phase for normal cells. Components of the G1 checkpoint, including retinoblastoma (Rb) protein, cyclin D1 and p16INK4a, are commonly altered in human malignancies, abrogating cell cycle control. Using immunohistochemistry, we examined 79 invasive transitional cell carcinomas of the urinary bladder treated by cystectomy, for loss of Rb or p16INK4a protein and for cyclin D1 overexpression. As p53 is also involved in cell cycle control, its expression was studied as well. Rb protein loss occurred in 23/79 cases (29%); it was inversely correlated with loss of p16INK4a, which occurred in 15/79 cases (19%). One biphenotypic case, with Rb+p16- and Rb-p16+ areas, was identified as well. Cyclin D1 was overexpressed in 21/79 carcinomas (27%), all of which retained Rb protein. Fifty of 79 tumours (63%) showed aberrant accumulation of p53 protein; p53 staining did not correlate with Rb, p16INK4a, or cyclin D1 status. Overall, 70% of bladder carcinomas showed abnormalities in one or more of the intrinsic proteins of the G1 checkpoint (Rb, p16INK4a and cyclin D1). Only 15% of all bladder carcinomas (12/79) showed a normal phenotype for all four proteins. In a multivariate survival analysis, cyclin D1 overexpression was linked to less aggressive disease and relatively favourable outcome. In our series, Rb, p16INK4a and p53 status did not reach statistical significance as prognostic factors. In conclusion, G1 restriction point defects can be identified in the majority of bladder carcinomas. Our findings support the hypothesis that cyclin D1 and p16INK4a can cooperate to dysregulate the cell cycle, but that loss of Rb protein abolishes the G1 checkpoint completely, removing any selective advantage for cells that alter additional cell cycle proteins.