Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.
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BACKGROUND: Normal human podocytes are terminally differentiated and quiescent cells. It is not known why podocytes fail to proliferate in response to most forms of injury. Proliferation is regulated by cell cycle proteins and their inhibitors. The Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) in general prevent proliferation by inhibiting cyclin-CDK complexes. In the current study, we determined the expression and possible role of specific CDK inhibitors in podocyte proliferation in human disease characterized by podocyte injury. METHODS: Immunostaining was performed for the CDK inhibitors p21, p27, and p57 and the proliferation marker Ki-67 on renal biopsies from patients with minimal change disease (MCD; N = 6), membranous glomerulopathy (MGN; N = 19), cellular variant of focal segmental glomerulosclerosis (FSGS; N = 12), collapsing glomerulopathy (CG; N = 9), and HIV-associated nephropathy (HIVAN; N = 16). Adult nephrectomy specimens without evidence of glomerular disease served as controls (N = 9). RESULTS: Normal quiescent podocytes express p27 and p57, but not p21. In diseases without podocyte proliferation (MCD, MGN), p21, p27, and p57 expression did not change. In contrast, there was a uniform decrease in p27 and p57 immunostaining in diseases with podocyte proliferation (cellular FSGS, CG, and HIVAN). This was accompanied by the de novo expression of p21 in podocytes. CONCLUSIONS: Our results show that podocyte quiescence may require the presence of the CDK inhibitors p27 and p57. In human glomerular diseases, a decrease in p27 and p57 may be permissive for the altered proliferative podocyte phenotype. p21 may have a multifactorial role in podocyte cell cycle regulation. (+info)
Mutation and expression of the p27KIP1 and p57KIP2 genes in human gastric cancer.
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Cyclin-dependent kinase inhibitors (CDKI) are negative regulators of cell cycle progression by binding the cyclin-CDK complex and inhibiting the CDK activity. Genetic alteration in the CDKI genes has been implicated for carcinogenesis. To test the genetic alteration in the p27 and p57 genes, KIP family CDKI genes, 30 gastric tumor-normal pairs and 8 gastric cancer cell lines were analyzed for mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). No mutation was detected in these genes although length polymorphisms in the proline-alanine repeat of the p57 gene were detected. When the p27 and p57 mRNAs were analyzed in gastric cancer cell lines by RT-PCR, the p27 mRNA was expressed considerably high in tumor cells but expression of the p57 mRNA was much low in gastric cancer cell lines compared to that of normal cells. The result suggests that inactivation of gene expression rather than mutations in the p57 gene accounts possibly for the involvement of this gene in tumorigenesis of gastric cancer. However, expression of the p27 gene seems to be essential for cell survival. (+info)
Disruption of imprinted genes at chromosome region 11p15.5 in paediatric rhabdomyosarcoma.
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Rhabdomyosarcomas are characterized by loss of heterozygosity (LOH) at chromosome region 11p15.5, a region known to contain several imprinted genes including insulin-like growth factor 2 (IGF2), H19, and p57(KIP2). We analyzed 48 primary tumour samples and found distinct genetic changes at 11p15.5 in alveolar and embryonal histological subtypes. LOH was a feature of embryonal tumours, but at a lower frequency than previous studies. Loss of imprinting (LOI) of the IGF2 gene was detected in 6 of 13 informative cases, all harbouring PAX3-FKHR or PAX7-FKHR fusion genes characteristic of alveolar histology. In contrast, H19 imprinting was maintained in 14 of 15 informative cases and the case with H19 LOI had maintenance of the IGF2 imprint indicating separate mechanisms controlling imprinting of IGF2 and H19. The adult promoter of IGF2, P1, was used in 5 of 14 tumours and its expression was unrelated to IGF2 imprinting status implying a further mechanism of altered IGF2 regulation. The putative tumour suppressor gene p57(KIP2) was expressed in 15 of 29 tumours and expression was unrelated to allele status. Moreover, in tumours with p57(KIP2) expression, there was no evidence for inactivating mutations, suggesting that p57(KIP2) is not a tumour suppressor in rhabdomyosarcoma. (+info)
Decreased expression of p57(KIP2)mRNA in human bladder cancer.
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To identify targets of genetic and epigenetic alterations on chromosome 11p15.5 in human bladder cancer, expression of the imprinted KIP2, IGF2 and H19 genes was studied by quantitative RT-PCR in 24 paired samples of urothelial carcinomas and morphologically normal mucosa obtained by cystectomy, and in bladder carcinoma cell lines. The most frequent alteration in tumour tissue was decreased expression of KIP2 identified in 9/24 (37%) specimens. Decreased IGF2 and H19 mRNA levels were found in five (21%) and three (13%) tumours, respectively. One tumour each overexpressed IGF2 and H19. Loss of H19 expression was only found associated with loss of KIP2 expression, whereas decreased expression of IGF2 mRNA occurred independently. Almost all bladder carcinoma cell lines showed significant changes in the expression of at least one gene with diminished expression of KIP2 mRNA as the most frequent alteration. IGF2 mRNA levels were diminished in several lines, but increased in others. The KIP2 gene could be an important target of genetic and epigenetic alterations in bladder cancer affecting the maternal chromosome 11p15.5. However, reminiscent of the situation in Wilms' tumours, expression of the IGF2 gene on the paternal chromosome can also be disturbed in bladder cancers. (+info)
Loss of imprinting and genetic alterations of the cyclin-dependent kinase inhibitor p57KIP2 gene in head and neck squamous cell carcinoma.
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The p57KIP2 is a maternally expressed and paternally imprinted cyclin-dependent kinase inhibitor located on chromosome 11p15.5. Because of its location, biochemical functions, and imprinting status, p57KIP2 has been considered a candidate tumor suppressor gene. To determine, for the first time, the involvement of this gene in the development of head and neck squamous carcinoma (HNSC), we analyzed the imprinting and expression status and loss of heterozygosity (LOH) within the p57KIP2 gene flanking loci on the 11p15.5 region in 64 primary untreated tumors. Of the 30 (47%) informative cases for this gene, loss of imprinting and LOH were noted in 4 (13%) and 10 tumors (33%), respectively. Analysis of the microsatellite markers flanking the p57KIP2 gene on chromosome 11p showed infrequent alterations at these loci. p57KIP2 was expressed in all tumors with LOH within and around the gene. Quantitative reverse transcription-PCR analysis showed elevated p57 mRNA expression in tumor with loss of imprinting. Sequencing analysis of exons 1 and 2 of the p57KIP gene failed to detect any mutations. Our data indicate: (a) infrequent genomic abnormalities at the p57KIP2 gene in HNSC; (b) leaky or incomplete imprinting of the paternal allele is associated with increased expression of this gene in a subset of tumors; and (c) minimal evidence for suppressor function for this gene in HNSC. (+info)
Expression of p57(KIP2) potently blocks the growth of human astrocytomas and induces cell senescence.
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Astrocytic tumors frequently exhibit defects in the expression or activity of proteins that control cell-cycle progression. Inhibition of kinase activity associated with cyclin/cyclin-dependent kinase co-complexes by cyclin-dependent kinase inhibitors is an important mechanism by which the effects of growth signals are down-regulated. We undertook the present study to determine the role of p57(KIP2) (p57) in human astrocytomas. We demonstrate here that whereas p57 is expressed in fetal brain tissue, specimens of astrocytomas of varying grade and permanent astrocytoma cell lines do not express p57, and do not contain mutations of the p57 gene by multiplex-heteroduplex analysis. However, the inducible expression of p57 in three well-characterized human astrocytoma cell lines (U343 MG-A, U87 MG, and U373 MG) using the tetracycline repressor system leads to a potent proliferative block in G(1) as determined by growth curve and flow cytometric analyses. After the induction of p57, retinoblastoma protein, p107, and E2F-1 levels diminish, and retinoblastoma protein is shifted to a hypophosphorylated form. Morphologically, p57-induced astrocytoma cells became large and flat with an expanded cytoplasm. The inducible expression of p57 leads to the accumulation of senescence-associated beta-galactosidase marker within all astrocytoma cell lines such that approximately 75% of cells were positive at 1 week after induction. Induction of p57 in U373 astrocytoma cells generated a small population of cells ( approximately 15%) that were nonviable, contained discrete nuclear fragments on Hoechst 33258 staining, and demonstrated ultrastructural features characteristic of apoptosis. Examination of bax and poly-(ADP ribose) polymerase levels showed no change in bax, but decreased expression of poly-(ADP ribose) polymerase after p57 induction in all astrocytoma cell lines. These data demonstrate that the proliferative block imposed by p57 on human astrocytoma cells results in changes in the expression of a number of cell cycle regulatory factors, cell morphology, and a strong stimulus to cell senescence. (+info)
p57(KIP2) is not mutated in hepatoblastoma but shows increased transcriptional activity in a comparative analysis of the three imprinted genes p57(KIP2), IGF2, and H19.
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Hepatoblastomas (HBs), representing malignant liver tumors of childhood, show frequent loss of heterozygosity (LOH) in the chromosomal region 11p15.5. This loss is of maternal origin suggesting the presence of a monoallelically expressed tumor suppressor gene in this region. p57(KIP2) (KIP2) located at 11p15.5 is predominantly expressed from the maternal allele and encodes a cyclin-dependent kinase inhibitor. We screened a series of 56 HB tumors and five HB cell lines for allelic loss (LOH) of the KIP2 locus by microsatellite analysis and KIP2 coding sequence mutations by single-strand conformation polymorphism analysis. Although LOH at the KIP2 locus occurred in 25% of the cases, no mutations were found. Analysis of KIP2 mRNA expression by competitive reverse transcriptase-polymerase chain reaction revealed up-regulation in nine of 12 HBs compared to matching liver samples. In contrast, mRNA levels of the putative suppressor gene H19 on 11p15.5 were decreased in 10 of 12 tumors, indicating that KIP2 and H19 are not co-regulated in HBs. IGF2 mRNA expression was increased in 11 of 12 HB samples. All HBs showed monoallelic KIP2 expression. However, the overexpression of KIP2 in HBs with maternal loss of 11p15.5 suggests a reactivation of the paternal allele in these cases. Overexpression of KIP2 in HBs argues against a role as a HB suppressor gene. (+info)
A potential role for p15(Ink4b) and p57(Kip2) in liver development.
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Hepatocytes undergo marked changes in proliferation during normal liver development. In order to elucidate the mechanism for these changes, we examined the ontogeny of expression for the known cyclin-dependent kinase inhibitors (CKIs), p15(Ink4b), p16(Ink4a), p18(Ink4c), p19(Ink4d), p21(Cip1), p27(Kip1) and p57(Kip2). All except p16(Ink4a) were expressed at some time between late gestation and adulthood. The mRNA and protein expression patterns for p15(Ink4b) and p57(Kip2) were consistent with a role for these CKIs in the regulation of hepatocyte proliferation. Specifically, p57(Kip2) may contribute to hepatocyte growth arrest that occurs in term fetuses, while p15(Ink4b) may contribute to the maintenance of adult hepatocytes in a quiescent state. These results assign a possible role to two CKIs not previously identified as involved in hepatocyte cell cycle control. (+info)