Cyclin D1 and Cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits. (73/1100)

BACKGROUND AND PURPOSE: The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We hypothesized that spinal motor neurons might be lost by programmed cell death and investigated a possible mechanism of neuronal death by detection of double-strand breaks in genomic DNA and immunohistochemical analysis for cyclin D1 and cyclin-dependent kinases (Cdk) 4. METHODS: We used a rabbit spinal cord ischemia model with a balloon catheter. Spinal cord was removed at 8 hours and 1, 2, and 7 days after 15 minutes of transient ischemia, and histological changes were studied with hematoxylin-eosin staining. In situ terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), DNA fragment with gel electrophoresis, Western blot analysis for cyclin D1 and Cdk4, and temporal profiles of cyclin D1 and Cdk4 immunoreactivity were investigated. RESULTS: Most motor neurons were preserved until 2 days but were selectively lost at 7 days of reperfusion. Immunocytochemistry showed positive TUNEL selectively at 2 days of reperfusion in spinal motor neuron nuclei. Typical ladders of oligonucleosomal DNA fragments were detected at 2 days of reperfusion. Immunoreactivity of cyclin D1 and Cdk4 proteins was induced selectively at 8 hours in motor neuron nuclei, which eventually died. CONCLUSIONS: These results indicate that induction of cyclin D1 and Cdk4 may be implicated in programmed cell death change after transient spinal cord ischemia in rabbits.  (+info)

The p16-cyclin D1/CDK4-pRb pathway and clinical outcome in epithelial ovarian cancer. (74/1100)

A significant positive association has been reported between p16 expression and clinical outcome for epithelial ovarian cancer patients. However, there is a reciprocal correlation between genetic alterations of single members of the p16-cyclin D1/CDK4-pRb pathway (G1 pathway). Simultaneous evaluation of these four elements may produce a better prognostic factor than p16 alone. We studied the prognostic significance of the G1 pathway in 59 epithelial ovarian cancer patients undergoing surgery and platinum-based chemotherapy by immunohistochemical technique. Abnormal expression of p16 or pRb was defined by negative nuclei staining, and that of CDK4 and cyclin D1 was defined by 50% nuclear staining. An abnormal G1 pathway was indicated in cases that have at least one abnormality among these four elements. Abnormal expression of p16, pRb, and cyclin D1/CDK4 was observed in 33.9, 3.4, and 15.3% of studied cases, respectively. Abnormal G1 pathway was detected in 49.2% (29 of 59) of all cases. The patients with normal G1 pathway tended to achieve a higher complete response rate (81.0%) to chemotherapy, compared with patients with abnormal G1 pathway (55.0%); however, there was no significant difference (P = 0.1001) between the two groups. Univariate analyses identified advanced stage [hazards ratio (HR), 3.665; P = 0.0218], histological low grade (HR, 3.625; P = 0.0066), and abnormal G1 pathway (HR, 2.935; P = 0.03) as prognostic factors for overall survival. The G1 pathway might help as a prognostic factor to select high-risk patients.  (+info)

The p16 status of tumor cell lines identifies small molecule inhibitors specific for cyclin-dependent kinase 4. (75/1100)

Loss of p16 functional activity leading to disruption of the p16/cyclin-dependent kinase (CDK) 4:cyclin D/retinoblastoma pathway is the most common event in human tumorigenesis, suggesting that compounds with CDK4 kinase inhibitory activity may be useful to regulate cancer cell growth. To identify such inhibitors, the 60 cancer cell lines of the National Cancer Institute drug screen panel were examined for p16 alterations (biallelic deletion, intragenic mutations, or absent p16 protein), and the growth-inhibitory activity of more than 50,000 compounds against these 60 cell lines was compared with their p16 status. One compound, 3-amino thioacridone (3-ATA; NSC 680434), whose growth-inhibitory activity correlated with the p16 status of the cell lines had an IC50 of 3.1 microM in a CDK4 kinase assay. In addition, four compounds structurally related to 3-ATA inhibited CDK4 kinase with IC50s ranging from 0.2-2.0 microM. All five of these compounds were less potent inhibitors of cell division cycle 2 and CDK2 kinases, with IC50s 30- to 500-fold higher than that for CDK4. ATP competition experiments demonstrated a noncompetitive mode of inhibition for 3-ATA (K(i) = 5.5 microM) and a linear mixed mode for benzothiadiazine (NSC 645787; K(i) = 0.73 microM). We have successfully demonstrated a novel approach to identify specific CDK4 kinase inhibitors that may selectively induce growth inhibition of p16-altered tumors.  (+info)

Deregulated expression of cyclin D1 overrides antimitogenic signals. (76/1100)

Several types of epithelial neoplasms exhibit high expression of transforming growth factor beta1 (TGFbeta-1), indicating that they have acquired tolerance to this normally growth inhibitory cytokine. Since cyclin D1 is expressed at high levels in murine skin tumors coincident with high levels of TGFbeta-1 expression, we hypothesized that cyclin D1 may override TGFbeta-1 induced growth arrest. We observed that in primary murine keratinocytes treated with TGFbeta-1, cyclin D1 is quickly suppressed at both the mRNA and protein level. Since changes in other cell cycle proteins occur at a later time during TGFbeta-1 treatment, the early suppression of cyclin D1 suggests that this gene is a critical target for TGFbeta-1 growth suppression. Using primary keratinocytes from transgenic mice that overexpress cyclin D1 (K5-D1 mice), we observed partial resistance to TGFbeta-1 growth inhibition. This resistance involves changes in the cyclin/cdk/inhibitor complexes rather than differences in expression of the TGFbeta receptors or signaling. Comparison of cdk associated kinase activity between wild-type and K5-D1 cells shows differential regulation. We conclude that deregulated cyclin D1 and subsequent alterations in cell cycle machinery provides keratinocytes the ability to at least partially override growth inhibitory signals.  (+info)

Retinoic acid-induced proliferation of lung alveolar epithelial cells is linked to p21(CIP1) downregulation. (77/1100)

Retinoids, including retinol and retinoic acid (RA) derivatives, have been shown to be involved in the processes of lung development as well as of lung repair after injury. Recently, we have provided evidence that RA could stimulate proliferation of lung alveolar type 2 epithelial cells (E. Nabeyrat, V. Besnard, S. Corroyer, V. Cazals, and A. Clement. Am. J. Physiol. Lung Cell. Mol. Physiol. 275: L71-L79, 1998). To gain some insight into the mechanisms involved in the mitogenic action of RA, we focused in the present study on the effects of RA on the expression of G(1) phase cyclins and their cell cycle-dependent kinases (Cdks). Experiments were performed with serum-deprived cells cultured in the absence and presence of RA. The results showed no effects of RA on the expression of either cyclins or Cdks. In contrast, RA treatment was found to prevent the decrease in cyclin E-Cdk2 activity observed when cells were growth arrested by serum deprivation. The observation that changes in cyclin E-Cdk2 activity were not associated with modifications in the amount of complexes formed led to the suggestion that the Cdk inhibitory protein (CKI) was involved. Study of the CKI p21(CIP1) revealed marked differences in its expression in the absence and presence of RA, with a dramatic downregulation observed in RA-treated cells. Interestingly, immunoprecipitation experiments provided evidence that the decreased levels of p21(CIP1) were associated with a reduced interaction of this CKI with cyclin E-Cdk2 complexes. These data together with previous results obtained in various situations of type 2 cell growth arrest emphasize the role of p21(CIP1) in the control of lung alveolar epithelial cell proliferation.  (+info)

Inhibitory effects of 1,3-diaminopropane, an ornithine decarboxylase inhibitor, on rat two-stage urinary bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine. (78/1100)

Overexpression of ornithine decarboxylase (ODC) has been shown to be characteristic of tumor development and progression in humans and experimental animals. Therefore, we have examined the effects of 1, 3-diaminopropane dihydrochloride (DAP), a potent inhibitor of ODC, on rat two-stage urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). In experiment 1 (36 weeks), 6-week-old F344 male rats were administered 0.05% BBN in drinking water for 4 weeks and then divided into four groups. Animals of groups 1 and 2 received basal diet and drinking water supplemented with or without DAP (2 g/l). Groups 3 and 4 were given diet containing 5% sodium L-ascorbate (NaAsA), a typical urinary bladder tumor promoter, and drinking water with or without DAP. Administration of DAP to group 1 significantly reduced tumor size, multiplicity and incidence, particularly of papillomas, when compared with group 2 values. DAP together with NaAsA (group 3) also decreased tumor size relative to the group 4 case. To determine the effects of DAP on the early stages of bladder carcinogenesis and its mechanisms, a similar protocol was conducted (experiment 2) with death after 20 weeks. DAP treatment caused complete inhibition (0% incidence) of papillary and/or nodular hyperplasia in group 1 but was without influence in group 3, as compared with the respective controls. Moreover, the ODC activity, bromodeoxyuridine labeling indices and mRNA expression levels of cyclin D1 in the urinary bladder mucosa, determined by northern blotting, were markedly lower in group 1 than in group 2, but values were comparable for both groups administered NaAsA. Assessment of mRNA expression levels of the angiogenic vascular endothelial growth factor suggested no involvement in the inhibitory effects of DAP on urinary bladder carcinogenesis. The results indicate that inhibition of ODC could reduce urinary bladder carcinogenesis in rats, particularly in the early stages, through antiproliferative mechanisms.  (+info)

Regulation of BRCA1 expression by the Rb-E2F pathway. (79/1100)

Inheritance of a mutant allele of the breast cancer susceptibility gene BRCA1 confers increased risk of developing breast and ovarian cancers. Likewise, inheritance of a mutant allele of the retinoblastoma susceptibility gene (RB1) results in the development of retinoblastoma and/or osteosarcoma, and both alleles are often mutated or inactivated in sporadic forms of these and other cancers. We now demonstrate that the product of the RB1 gene, Rb, regulates the expression of the murine Brca1 and human BRCA1 genes through its ability to modulate E2F transcriptional activity. The Brca1 gene is identified as an in vivo target of E2F1 in a transgenic mouse model. The Brca1 promoter contains E2F DNA-binding sites that mediate transcriptional activation by E2F1 and repression by Rb. Moreover, ectopic expression of cyclin D1 and Cdk4 can stimulate the Brca1 promoter in an E2F-dependent manner, and this is inhibited by coexpression of the p16(INK4a) cyclin-dependent kinase inhibitor. The human BRCA1 promoter also contains a conserved E2F site and is similarly regulated by E2F1 and Rb. This functional link between the BRCA1 and Rb tumor suppressors may provide insight into the mechanism by which BRCA1 inactivation contributes to cancer development.  (+info)

Genetic profile of gliosarcomas. (80/1100)

There are distinct genetic pathways leading to the glioblastoma, the most malignant astrocytic brain tumor. Primary (de novo) glioblastomas develop in older patients and are characterized by epidermal growth factor (EGF) receptor amplification/overexpression, p16 deletion, and PTEN mutations, whereas secondary glioblastomas that progressed from low-grade or anaplastic astrocytoma develop in younger patients and frequently contain p53 mutations. In this study, we assessed the genetic profile of gliosarcoma, a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas displaying glial and mesenchymal differentiation. Single-strand conformation polymorphism followed by direct DNA sequencing revealed p53 mutations in five of 19 gliosarcomas (26%) and PTEN mutations in seven cases (37%). Homozygous p16 deletion was detected by differential polymerase chain reaction in seven (37%) gliosarcomas. The overall incidence of alterations in the Rb pathway (p16 deletion, CDK4 amplification, or loss of pRb immunoreactivity) was 53%, and these changes were mutually exclusive. Coamplification of CDK4 and MDM2 was detected in one gliosarcoma. None of the gliosarcomas showed amplification or overexpression of the EGF receptor. Thus gliosarcomas exhibit a genetic profile similar to that of primary (de novo) glioblastomas, except for the absence of EGFR amplification/overexpression. Identical PTEN mutations in the gliomatous and sarcomatous tumor components were found in two cases. Other biopsies contained p16 deletions, an identical p53 mutation, or coamplification of MDM2 and CDK4 in both tumor areas. This strongly supports the concept of a monoclonal origin of gliosarcomas and an evolution of the sarcomatous component due to aberrant mesenchymal differentiation in a highly malignant astrocytic neoplasm.  (+info)