The yeast ser/thr phosphatases sit4 and ppz1 play opposite roles in regulation of the cell cycle.
(9/5371)
Yeast cells overexpressing the Ser/Thr protein phosphatase Ppz1 display a slow-growth phenotype. These cells recover slowly from alpha-factor or nutrient depletion-induced G1 arrest, showing a considerable delay in bud emergence as well as in the expression of the G1 cyclins Cln2 and Clb5. Therefore, an excess of the Ppz1 phosphatase interferes with the normal transition from G1 to S phase. The growth defect is rescued by overexpression of the HAL3/SIS2 gene, encoding a negative regulator of Ppz1. High-copy-number expression of HAL3/SIS2 has been reported to improve cell growth and to increase expression of G1 cyclins in sit4 phosphatase mutants. We show here that the described effects of HAL3/SIS2 on sit4 mutants are fully mediated by the Ppz1 phosphatase. The growth defect caused by overexpression of PPZ1 is intensified in strains with low G1 cyclin levels (such as bck2Delta or cln3Delta mutants), whereas mutation of PPZ1 rescues the synthetic lethal phenotype of sit4 cln3 mutants. These results reveal a role for Ppz1 as a regulatory component of the yeast cell cycle, reinforce the notion that Hal3/Sis2 serves as a negative modulator of the biological functions of Ppz1, and indicate that the Sit4 and Ppz1 Ser/Thr phosphatases play opposite roles in control of the G1/S transition. (+info)
Immunohistochemical expression of mdm2 and p21WAF1 in invasive cervical cancer: correlation with p53 protein and high risk HPV infection.
(10/5371)
AIM: To investigate the immunocytochemical staining pattern of mdm2 and p21WAF1 proteins in invasive cervical cancer and to determine its relation with the expression of p53 and with the high risk HPV infection. METHODS: Immunocytochemistry for p53, mdm2, and p21WAF1 was performed in 31 paraffin embedded sections of invasive cervical cancer. The results were assessed by image analysis, evaluating for each protein the optical density of the immunostained area, scored as percentage of the total nuclear area. The presence of high risk human papillomavirus (HPV) infection was detected by using the polymerase chain reaction. RESULTS: Immunostaining for both mdm2 and p21WAF1 was correlated with p53 expression; however, the correlation between p53 and mdm2 (R = 0.49; p < 0.01) was more significant than between p53 and p21WAF1 (R = 0.31; p < 0.05); the less stringent correlation between p53 and p21WAF1 might reflect the p53 independent mechanisms of p21WAF1 induction. Similar average levels of p53, mdm2, and p21WAF1 immunostaining were found in the presence or absence of high risk HPV-DNA, without significant differences between the two groups. CONCLUSIONS: These data suggest that mdm2 and p21WAF1 proteins are expressed in invasive cervical cancer and that their immunocytochemical staining pattern is not abrogated by the presence of high risk HPV genomic sequences. (+info)
Interleukin-6 dependent induction of the cyclin dependent kinase inhibitor p21WAF1/CIP1 is lost during progression of human malignant melanoma.
(11/5371)
Human melanoma cell lines derived from early stage primary tumors are particularly sensitive to growth arrest induced by interleukin-6 (IL-6). This response is lost in cell lines derived from advanced lesions, a phenomenon which may contribute to tumor aggressiveness. We sought to determine whether resistance to growth inhibition by IL-6 can be explained by oncogenic alterations in cell cycle regulators or relevant components of intracellular signaling. Our results show that IL-6 treatment of early stage melanoma cell lines caused G1 arrest, which could not be explained by changes in levels of G1 cyclins (D1, E), cdks (cdk4, cdk2) or by loss of cyclin/cdk complex formation. Instead, IL-6 caused a marked induction of the cdk inhibitor p21WAF1/CIP1 in three different IL-6 sensitive cell lines, two of which also showed a marked accumulation of the cdk inhibitor p27Kip1. In contrast, IL-6 failed to induce p21WAF1/CIP1 transcript and did not increase p21WAF1/CIP1 or p27kip1 proteins in any of the resistant lines. In fact, of five IL-6 resistant cell lines, only two expressed detectable levels of p21WAF1/CIP1 mRNA and protein, while in three other lines, p21WAF1/CIP1 was undetectable. IL-6 dependent upregulation of p21WAF1/CIP1 was associated with binding of both STAT3 and STAT1 to the p21WAF1/CIP1 promoter. Surprisingly, however, IL-6 stimulated STAT binding to this promoter in both sensitive and resistant cell lines (with one exception), suggesting that gross deregulation of this event is not the unifying cause of the defect in p21WAF1/CIP1 induction in IL-6 resistant cells. In somatic cell hybrids of IL-6 sensitive and resistant cell lines, the resistant phenotype was dominant and IL-6 failed to induce p21WAF1/CIP1. Thus, our results suggest that in early stage human melanoma cells, IL-6 induced growth inhibition involves induction of p21WAF1/CIP1 which is lost in the course of tumor progression presumably as a result of a dominant oncogenic event. (+info)
Differential regulation of p21waf-1/cip-1 and Mdm2 by etoposide: etoposide inhibits the p53-Mdm2 autoregulatory feedback loop.
(12/5371)
The Mdm2 protein is frequently overexpressed in human non-seminomatous germ cell tumours and transitional carcinoma of the bladder where it may contribute to tolerance of wtp53. Mdm2 forms an autoregulatory feedback loop with p53; the Mdm2 gene is responsive to transactivation by p53 and once synthesized the Mdm2 protein terminates the p53 response. We show here that the topoisomerase poison etoposide, like ultra violet irradiation, inhibits Mdm2 synthesis. Cytotoxic concentrations of etoposide (IC90 for > 3 h) result in inhibition of Mdm2 induction at both the RNA and protein level. Rapid apoptosis ensues. Global transcription is not inhibited: p21waf-1/cip1 and GADD45 expression increase in a dose dependent manner. Inhibition of Mdm2 synthesis depends on the continuous presence of etoposide, suggesting the DNA damage may prevent transcription. Downregulation of Mdm2 transcript occurs in cells expressing HPV16-E6 suggesting that inhibition of Mdm2 transcription is p53-independent. When cells are -treated with a pulse (1 h) of etoposide and reincubated in drug free medium, Mdm2 synthesis commences immediately after damage is repaired (3 h) and the p53 response is attenuated. Induction of apoptosis and loss of clonogenicity are 3-5-fold lower under pulse treatment conditions. This is the first observation of inhibition of Mdm2 transcription following treatment with topoisomerase (topo II) poisons, a feature that may be useful in tumour types where p53 is tolerated by overexpression of Mdm2. (+info)
Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases.
(13/5371)
Phosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II is important for basal transcriptional processes in vivo and for cell viability. Several kinases, including certain cyclin-dependent kinases, can phosphorylate this substrate in vitro. It has been proposed that differential CTD phosphorylation by different kinases may regulate distinct transcriptional processes. We have found that two of these kinases, cyclin C/CDK8 and cyclin H/CDK7/p36, can specifically phosphorylate distinct residues in recombinant CTD substrates. This difference in specificity may be largely due to their varying ability to phosphorylate lysine-substituted heptapeptide repeats within the CTD, since they phosphorylate the same residue in CTD consensus heptapeptide repeats. Furthermore, this substrate specificity is reflected in vivo where cyclin C/ CDK8 and cyclin H/CDK7/p36 can differentially phosphorylate an endogenous RNA polymerase II substrate. Several small-molecule kinase inhibitors have different specificities for these related kinases, indicating that these enzymes have diverse active-site conformations. These results suggest that cyclin C/CDK8 and cyclin H/CDK7/p36 are physically distinct enzymes that may have unique roles in transcriptional regulation mediated by their phosphorylation of specific sites on RNA polymerase II. (+info)
Circadian variation in the expression of cell-cycle proteins in human oral epithelium.
(14/5371)
At the tissue level, there is experimental and clinical data to suggest a cytokinetic coordination of the cell cycle with a greater proportion of cycling cells entering S-phase and mitosis at specific times of the day. The association of certain cell-cycle proteins with defined events in the cell cycle is well established and may be used to study the timing of cell-cycle phases over 24 hours. In this study oral mucosal biopsies were obtained from six normal human volunteers at 4-hour intervals, six times over 24 hours. Using immunohistochemistry, the number of positive cells expressing the proteins p53, cyclin-E, cyclin-A, cyclin-B1, and Ki-67 was determined for each biopsy and expressed as the number of positive cells per mm of basement membrane. We found a statistically significant circadian variation in the nuclear expression of all of these proteins with the high point of expression for p53 at 10:56 hours, cyclin-E at 14:59 hours, cyclin-A at 16:09 hours, cyclin-B1 at 21:13 hours, and Ki-67 at 02:50 hours. The circadian variation in the nuclear expression of cyclins-E (G1/S phase), -A (G2-phase), and -B1 (M-phase) with a normal physiological progression over time suggests a statistically significant circadian variation in oral epithelial cell proliferation. The finding of a circadian variation in the nuclear expression of p53 protein corresponding to late G1 is novel. This information has clinical implications regarding the timing of chemotherapy and radiotherapy. (+info)
Detection of Kaposi's sarcoma herpesvirus DNA sequences in multiple myeloma bone marrow stromal cells.
(15/5371)
Whether Kaposi's sarcoma herpesvirus (KSHV) is associated with multiple myeloma (MM) remains controversial. We assayed for KSHV DNA sequences in long-term bone marrow stromal cells (BMSCs) from 26 patients with MM and 4 normal donors. Polymerase chain reaction (PCR) using primers which amplify a KSHV gene sequence to yield a 233-bp fragment (KS330233 within open reading frame 26) was negative in all cases. Aliquots of these PCR products were used as templates in subsequent nested PCR, with primers that amplify a 186-bp product internal to KS330233. BMSCs from 24 of 26 (92%) patients with MM and 1 of 4 normal donors were KSHV PCR+. DNA sequence analyses showed interpatient specific mutations (2 to 3 bp). Both Southern blot and sequence analyses confirmed the specificity of PCR results. The presence of the KSHV gene sequences was further confirmed by amplifying T 1.1 (open reading frame [ORF] K7) and viral cyclin D (ORF 72), two other domains within the KSHV genome. Immunohistochemical studies of KSHV PCR+ MM BMSCs demonstrate expression of dendritic cell (DC) lineage markers (CD68, CD83, and fascin). Serological studies for the presence of KSHV lytic or latent antibodies were performed using sera from 53 MM patients, 12 normal donors, and 5 human immunodeficiency virus (HIV)/KSHV+ patients. No lytic or latent antibodies were present in sera from either MM patients or normal donors. Taken together, these findings show that KSHV DNA sequences are detectable in BMSCs from the majority of MM patients, but that serologic responses to KSHV are not present. Ongoing studies are defining whether the lack of antibody response is caused by the absence of ongoing infection, the presence of a novel viral strain associated with MM, or underlying immunodeficiency in these patients. (+info)
Activation and repression of p21(WAF1/CIP1) transcription by RB binding proteins.
(16/5371)
The Cdk inhibitor p21(WAF1/CIP1) is a negative regulator of the cell cycle, although its expression is induced by a number of mitogens that promote cell proliferation. We have found that E2F1 and E2F3, transcription factors that activate genes required for cell cycle progression, are strong activators of the p21 promoter. In contrast, HBP1 (HMG-box protein-1), a novel retinoblastoma protein-binding protein, can repress the p21 promoter and inhibit induction of p21 expression by E2F. Both E2Fs and HBP1 regulate p21 transcription through cis-acting elements located between nucleotides -119 to +16 of the p21 promoter and the DNA binding domains of each of these proteins are required for activity. Sequences between -119 and -60 basepairs containing four Sp1 consensus elements and two noncanonical E2F binding sites are of major importance for E2F activation, although E2F1 and E2F3 differ in the extent of their ability to activate expression when this segment is deleted. The opposing effects of E2Fs and HBP1 on p21 promoter activity suggest that interplay between these factors may determine the level of p21 transcription in vivo. (+info)