Inhibition Of Cyclin Research Articles

Magnesium in cell proliferation and differentiation.

Compelling evidence shows that magnesium (Mg) content is directly correlated to proliferation in normal cells as Mg stimulates DNA and protein synthesis. Some data have demonstrated that upon mitogenic stimuli normal cell are able to increase their intracellular Mg content, likely by activating Mg influx. Mg deprivation, in turn, induces inhibition of DNA and protein synthesis thus promoting growth arrest. From a mechanistic viewpoint, Mg deprivation may influence cell cycle control by upregulating the cyclin inhibitor p27Kip1 thus influencing cyclin E-dependent kinases. In many neoplastic cells, Mg is higher than in normal counterparts and this high Mg is maintained also against concentration gradient. Moderate vs. severe and acute vs chronic effect of Mg deprivation must be distinguished: severe Mg deprivation causes growth arrest also in tumor cells, while chronic Mg deprivation leads to an "adaptation" of tumor cells both to growth rate and Mg content. In tumor cells deranged Mg content and distribution is likely due to an inhibition of Mg efflux via the Na-Mg antiport. When differentiation process is induced by receptor mediated stimuli such as IFN-alpha and ATP, decrease of cell Mg content accompanies with activation of Mg efflux. Transformed cells may thus display high growth rate also because they retain a large amount of Mg. On their whole, these data strongly suggest that regulation of intracellular Mg availability parallels the molecular control of cell proliferation, and maybe also cell differentiation and death.

An indirect role for cyclin B‐Cdc2 in inducing chromosome condensation in Xenopus egg extracts*

We have studied the cytoplasmic mechanism that induces metaphase chromosome condensation in cell‐free Xenopus egg extracts. To analyze the mechanism responsible for inducing chromosome condensation separately from those responsible for sperm chromatin remodeling and nuclear envelope disassembly, we used Xenopus sperm chromatin that had already been remodeled to nucleosomal chromatin by incubating demembranated sperm with egg extracts added with lysolecithin. We found that inhibition of cyclin B‐Cdc2 with butyrolactone I abolished chromosome condensation of the remodeled sperm chromatin by M‐phase egg extracts, but incubation of the chromatin with active cyclin B‐Cdc2 alone did not induce chromosome condensation, indicating a requirement for cytoplasmic factor(s) in addition to cyclin B‐Cdc2 for the induction of chromosome condensation. We further demonstrated that if the cyclin B‐Cdc2‐dependent phosphorylation state was protected against dephosphorylation by a preincubation of M‐phase extracts with ATP‐γ‐S, chromosome condensation and phosphorylation of chromosomal histone H1 occurred even when extracts were depleted of cyclin B‐Cdc2 activity. The chromosome condensation seen in the absence of cyclin B‐Cdc2 was completely inhibited with another protein kinase inhibitor, 6‐dimethylaminopurine, implying that a protein kinase other than cyclin B‐Cdc2 was involved in the induction of chromosome condensation. These results strongly suggest that a cyclin B‐Cdc2‐dependent protein kinase cascade is involved in inducing chromosome condensation and the phosphorylation of chromosomal histone H1.

Effect of wortmannin and 3-AB on G2 phase arrest and transient failure to activate MPF induced by ionizing irradiation

Active mitosis promoting factor (MPF) composed of p34odc2 with cyclin B1 is required for the cell cycle transition from G2 to M phase. Recent studies have demonstrated that ionizing radiation (IR) exposures associated with inhibition of p34odc2 activity was the mechanism of G2 arrest. At low dose, IR causes transient failure to dephosphorylate p34odc2 instead of suppression of cyclin B or MPF formation of cyclin B with p34odc2 complex. But the signaling events that regulate p34odc2 in irradiated cells remain unclear. This note demonstrates that PI3 kinase family inhibitor wortmannin (wort) and PARP specific inhibitor 3-AB can reduce the G2 arrest induced by 2Gy γ-ray. Immunoprecipitation studies demonstrate that wort and 3-AB can facilitate dephosphorylation of p34odc2 in G2 phase arrest induced by radiation. These findings suggest that wort sensitive pathway and PARP may be involved in initiating the signal transduction of G2 phase arrest caused by IR.

Induction of cell cycle arrest by the endogenous product of lipid peroxidation, malondialdehyde.

We have investigated the effect of the endogenous genotoxin malondialdehyde (MDA) on cell cycle kinetics and the expression and biochemical activity of several cell cycle regulatory proteins. MDA treatment of two human cell lines (RKO and H1299) resulted in a 3- to 6-fold elevation in the levels of the major detectable MDA-DNA adduct, M1G-dR. The increase in M1G-dR was accompanied by irreversible cell cycle arrest, elevation in p53 and p21 protein levels, and inhibition of cyclin E- and cyclin B-associated kinase activities. The decrease in cyclin E- and cyclin B-dependent kinase activities was caused by increased p21 and decreased cdc2 levels, respectively. Comparable levels of p21 induction were observed in RKO (wild-type p53) and H1299 (p53-null) cells. Thus, MDA was able to engage cell cycle checkpoint function in human cell lines when used at concentrations that produce M1G-dR levels of the same magnitude found in human tissues.

Molecular basis for the lack of T cell proliferation induced by an altered peptide ligand.

In this report, we explore the mechanisms underlying cell cycle progression in T cells stimulated with an altered peptide ligand (APL) versus wild-type peptide. APL stimulation did not induce proliferation compared to wild-type peptide stimulation. To determine the point at which cell cycle progression is blocked, we have examined molecules responsible for regulating the retinoblastoma tumor suppressor gene product, pRb, which in its active state prevents G1/S progression. The majority of cells stimulated with an APL did not progress beyond G1; however, a small population did make the G1/S transition. These few cells passed the late G1 restriction point, divided and subsequently arrested at the next G1 phase. The lack of sustained signaling events following stimulation with an APL failed to induce cyclin E:cdk2 activity, a regulator which hyper-phosphorylates and inactivates pRb. Exogenous IL-2 addition did not compensate for the lack of proliferation following APL stimulation. Furthermore, the inability of the cells to enter S phase during partial T cell activation cannot be accounted for by p27Kip1 inhibition of cyclin E:cdk2 complexes. Upon APL stimulation, an increase in association of p27Kip1 with cyclin E:cdk2 complex was not observed, suggesting that instead, decreased cyclin E:cdk complex formation might contribute to the failure to progress from G1/S. Therefore, while for a majority of cells, wild-type stimulation results in cell cycle progression, APL stimulation is not sufficient to drive cells beyond G1.

Comparative Phenotypic Studies of Duct Epithelial Cell Lines Derived from Normal Human Pancreas and Pancreatic Carcinoma

We have investigated the mRNA/protein expression of several tyrosine kinase receptors, growth factors, and p16INK4A cyclin inhibitor in cell lines derived from normal human pancreatic duct epithelium (HPDE) and compared them with those of five pancreatic ductal carcinoma cell lines. Cultured HPDE cells express low levels of epidermal growth factor receptor (EGFR), erbB2, transforming growth factor (TGF)-alpha, Met/hepatocyte growth factor receptor (HGFR), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF). They also expressed high levels of amphiregulin but did not express EGF and cripto. The expression levels were similar in primary normal HPDE cells and those expressing transfected E6E7 genes of human papilloma virus-16, but their immortalization appeared to enhance the expression of EGFR and Met/HGFR. In comparison, pancreatic carcinoma cell lines commonly demonstrated overexpression of EGFR, erbB2, TGF-alpha, Met/HGFR, VEGF, and KGF, but they consistently showed marked down-regulation of amphiregulin mRNA expression. In contrast to all carcinoma cell lines that showed deletions of the p16 gene, HPDE cells consistently demonstrated normal p16 genotype and its mRNA expression. This is the first report that compares the phenotypic expression of cultured pancreatic ductal carcinoma cells with epithelial cell lines derived from normal human pancreatic ducts. The findings confirm that malignant transformation of human pancreatic duct cells commonly results in a deregulation of expression of various growth factors and receptors.

INDUCTION OF THE CYCLINE INHIBITOR p21 EXPRESSION BY CYCLOSPORINE: A POSSIBLE MECHANISM FOR ITS ANTIPROLIFERATIVE EFFECTS

674 The mechanism(s) of cyclosporine's (CsA) efficacy has been traditionally explained on the basis of inhibition of IL-2 and more recently, the induction TGF-β. Both the processes involve the inhibition of cell cycle progression. The cell cycle is controlled by cyclins, regulated by their association with cyclin-dependent kinases (cdks) which promote cell division. cdks are inhibited by selective cdk inhibitors, with prevent the cell from entering the cell-cycle. The most studied and significant cell cyclin inhibitor is p21. In this study, we hypothesized that the efficacy of CsA might be due to the hyperinduction of p21. To examine this hypothesis, we studied the effect of CsA on p21 gene and protein expression, we also examined the effect of CsA on the promoter activity of p21 gene. The expression of p21 gene and protein was studied in human lung adenocarcinoma cell line (A549 cells) cultured with or without CsA. RNA and cell lysates from these cells were prepared, mRNA expression for p21 and β-actin was studied using RT-PCR and p21 protein expression was studied by western blot analysis using anti-p21 monoclonal antibody and p21 control. The ethidium bromide stained bands were scanned and the expression of p21 gene was expressed relative to that of β-actin gene. Fig 1A shows a representative of three experiments which indicated a statistically significant (p=0.05) induction of the p21 gene by CsA (CsA treated 0.37 ± .02 vs 0.12 ± 0.05 untreated).Fig. 1B demonstrate the hyperexpression of p21 protein in the cell lysates of A-549 cells treated with CsA as compared to untreated cells. The stimulation of p21 promoter activity by CsA was studied in A-549 cells transfected with different concentrations (1.5-2.5 µg) of p21-luc plasmid DNA. Fig. 2 demonstrates the stimulation of the promoter activity of p21 by CsA. These results indicate that CsA induces p21 gene and protein and also stimulates its promoter activity. Interestingly, TGF-β has also been shown to induce p21 gene expression and stimulate its promoter activity. This data may suggest the possibility of another mediator for the anti-proliferative of CsA.Fig. 1A: Induction of p21 gene by CsAFig. 1B: Induction of p21 protein by CsAFig. 2: CsA induces the promoter activity of p21 gene

INDUCTION OF THE CYCLINE INHIBITOR p21 EXPRESSION BY CYCLOSPORINE: A POSSIBLE MECHANISM FOR ITS ANTIPROLIFERATIVE EFFECTS.

674 The mechanism(s) of cyclosporine's (CsA) efficacy has been traditionally explained on the basis of inhibition of IL-2 and more recently, the induction TGF-β. Both the processes involve the inhibition of cell cycle progression. The cell cycle is controlled by cyclins, regulated by their association with cyclin-dependent kinases (cdks) which promote cell division. cdks are inhibited by selective cdk inhibitors, which prevent the cell from entering the cell-cycle. The most studied and significant cell cyclin inhibitor is p21. In this study, we hypothesized that the efficacy of CsA might be due to the hyperinduction of p21. To examine this hypothesis, we studied the effect of CsA on p21 gene and protein expression, we also examined the effect of CsA on the promoter activity of p21 gene. The expression of p21 gene and protein was studied in human lung adenocarcinoma cell line (A549 cells) cultured with or without CsA. RNA and cell lysates from these cells were prepared, mRNA expression for p21 and β-actin was studied using RT-PCR and p21 protein expression was studied by western blot analysis using anti-p21 monoclonal antibody and p21 control. The ethidium bromide stained bands were scanned and the expression of p21 gene was expressed relative to that of β-actin gene. Fig 1A shows a representative of three experiments which indicated a statistically significant (p=0.05) induction of the p21 gene by CsA (CsA treated 0.37 ±.02 vs 0.12 ± 0.05 untreated).Fig. 1B demonstrate the hyperexpression of p21 protein in the cell lysates of A-549 cells treated with CsA as compared to untreated cells. The stimulation of p21 promoter activity by CsA was studied in A-549 cells transfected with different concentrations (1.5-2.5 μg) of p21-luc plasmid DNA. Fig. 2 demonstrates the stimulation of the promoter activity of p21 by CsA. These results indicate that CsA induces p21 gene and protein and also stimulates its promoter activity. Interestingly, TGF-β has also been shown to induce p21 gene expression and stimulate its promoter activity. This data may suggest the possibility of another mediator for the anti-proliferative of CsA.Fig. 1A Induction of p21 gene by CsAFig. 1B Induction of p21 protein by CsAFig.2 CsA induces the promoter activity of p21 gene

Myc and the cell cycle.

Ectopic expression of the c-Myc oncoprotein prevents cell cycle arrest in response to growth-inhibitory signals, differentiation stimuli, or mitogen withdrawal. Moreover, Myc activation in quiescent cells is sufficient to induce cell cycle entry in the absence of growth factors. Thus, Myc transduces a potent mitogenic stimulus but, concomitantly, induces apoptosis in the absence of survival factors. We review here recent progress in our understanding of the molecular mechanisms linking Myc activity to cell cycle control. Myc is a positive regulator of G1-specific cyclin-dependent kinases (CDKs) and, in particular, of cyclin E/CDK2 complexes. Cyclin D/CDK4 and CDK6 may conceivably also be activated by Myc, but the circumstances in which this occurs remain to be explored. Myc acts via at least three distinct pathways which can enhance CDK function: (1) functional inactivation of the CDK inhibitor p27Kip1 and probably also of p21Cip1 and p57Kip2, (2) induction of the CDK-activating phosphatase Cdc25A and (3) - in an ill understood and most likely indirect way - deregulation of cyclin E expression. Constitutive expression of either Myc or cyclin E can prevent growth arrest by p16INK4a (an inhibitor of cyclin D/CDK4, but not of cyclin E/CDK2). In cells, p16INK4a inhibits phosphorylation, and thus induces activation of the Retinoblastoma-family proteins (pRb, p107 and p130). Surprisingly, this effect of p16 is not altered in the presence of Myc or cyclin E. Thus, Myc and cyclin E/CDK2 activity unlink activation of p16 and pRb from growth arrest. Finally, Myc may itself be a functional target of cyclin D/CDK4 through its direct interaction with p107. We discuss how the effects of Myc on cell cycle control may relate to its oncogenic activity, and in particular to its ability to cooperate with activated Ras oncoproteins.

Cellular factors may enable squamous carcinoma cells to overcome TGF beta-mediated repression of CDK2 activity.

Cell lines developed from head and neck squamous cell carcinomas exhibit variable responses to the negative regulatory effects of transforming growth factor beta (TGF beta) on cell growth. To analyse the effects of TGF beta on regulators of cell cycle progression, we characterised cell lines derived from head and neck squamous cell carcinoma (HNSCC) for their biological sensitivities to TGF beta, growth inhibition, then examined the effects of TGF beta treatment on the expression and activity of cyclin dependent kinases (CDKs) and inhibitors of these kinases. Western blot analysis of cell lysates from untreated or TGF beta-treated cultures showed no alterations in expression of CDK2, CDK4, CDK6 or cyclin E in cell lines which were either sensitive (HaCaT, HN6) or refractory (HN12, HN30) to the growth-inhibitory effects of TGF beta. However, treatment of cells with TGF beta resulted in a several fold increase in cellular levels of p21 (WAF1/Cip1), irrespective of biological response. Immune complex in vitro kinase assays demonstrated that the activity of CDK2 was inhibited by exposure to ligand in each case, confirming that a TGF beta signalling pathway which regulates kinase activity was intact in these cell lines. The data suggest that cellular factors expressed in HN12 and HN30 enable these cells to override TGF beta-mediated inhibition of CDK2 activity and allow cell cycle progression. This may represent an important mechanism which allows cells to evade growth arrest during malignant progression.

Chromosome association of minichromosome maintenance proteins in Drosophila mitotic cycles.

Minichromosome maintenance (MCM) proteins are essential DNA replication factors conserved among eukaryotes. MCMs cycle between chromatin bound and dissociated states during each cell cycle. Their absence on chromatin is thought to contribute to the inability of a G2 nucleus to replicate DNA. Passage through mitosis restores the ability of MCMs to bind chromatin and the ability to replicate DNA. In Drosophila early embryonic cell cycles, which lack a G1 phase, MCMs reassociate with condensed chromosomes toward the end of mitosis. To explore the coupling between mitosis and MCM-chromatin interaction, we tested whether this reassociation requires mitotic degradation of cyclins. Arrest of mitosis by induced expression of nondegradable forms of cyclins A and/or B showed that reassociation of MCMs to chromatin requires cyclin A destruction but not cyclin B destruction. In contrast to the earlier mitoses, mitosis 16 (M16) is followed by G1, and MCMs do not reassociate with chromatin at the end of M16. dacapo mutant embryos lack an inhibitor of cyclin E, do not enter G1 quiescence after M16, and show mitotic reassociation of MCM proteins. We propose that cyclin E, inhibited by Dacapo in M16, promotes chromosome binding of MCMs. We suggest that cyclins have both positive and negative roles in controlling MCM-chromatin association.

Cyclin D1 and p16INK4A are preferentially expressed in immature and mature myeloma cells, respectively.

Myeloma cells consist of immature, intermediate and mature cells with respect to expression of VLA-5 (CD49e) and MPC-1 adhesion molecules. VLA-5(-)MPC-1(-) immature myeloma cells respond to interleukin 6 (IL-6) to proliferate in vitro. but VLA-5+MPC-1+ mature myeloma cells have almost no proliferative activity with higher secretory activity of M-protein in vitro. In order to further clarify the biological differences between these immature and mature myeloma cells, we examined survival of these cells with or without IL-6 in vitro, and investigated the underlying mechanism of the proliferative or non-proliferative character of these cells by examining expression of cell cycle regulators such as cyclin D1 and inhibitors for cyclin-dependent kinase (Cdk), p16INK4A, p21CIP1 and p27KIP1 by RT-PCR and immunohistochemistry. In vitro survival of these myeloma cells was examined by flow cytometric quantification of fluorescein diacetate (FDA) and propidium iodide (PI) staining. Immature myeloma cells rapidly entered apoptosis without IL-6, but mature myeloma cells could survive without IL-6 as well as normal mature plasma cells. Immature myeloma cells as well as myeloma cell lines expressed cyclin D1 mRNA and protein, but not any Cdk inhibitors. On the other hand, mature myeloma cells did not express cyclin D1 but expressed p16, not p21 or p27, as well as normal mature plasma cells. Therefore these results show that immature myeloma cells constitutively express cyclin D1 and can proliferate, and mature myeloma cells as well as normal mature plasma cells preferentially express p16 and can survive for a long time without proliferation.

Inhibition of cyclin D expression in human breast carcinoma cells by retinoids in vitro.

Transfection and transgenic mouse experiments supported an oncogenic role for cyclin D1 in breast cancer. We recently reported that noninvasive carcinoma in situ lesions of the human breast overexpress cyclin D, suggesting that this molecular event may represent a valuable target for chemoprevention. The purpose of the present series of investigations was to identify agents which could reduce the cyclin D expression of breast cells. We report that 9-cis retinoic acid (9-cis RA) and all trans retinoic acid (tRA) inhibited the cyclin D1 and D3 expression levels of human MCF-7, ZR-75 and T-47D breast carcinoma cells in vitro. Where detectable, similar trends were observed in the immortalized, HBL-100 and MCF-10A breast cell lines. Cyclin D2 was undetectable. The effect of retinoids was both dose- and time-dependent, and correlated with altered cell cycle kinetics and proliferative status. Retinoids were also found to inhibit the expression levels of other cell cycle related proteins, including Cdk2 and Cdk4, resulting in lower kinase activities. In contrast to other breast prevention studies, no synergistic effect was observed with retinoids and tamoxifen. The data indicate that retinoids can potently reduce cyclin D expression levels in a variety of breast cell lines in vitro, and suggest further consideration of this mechanism for the chemoprevention of breast cancer.

Features of Macrophage Differentiation Induced by p19INK4d, a Specific Inhibitor of Cyclin D–Dependent Kinases

The mitogen-dependent induction of cyclin D–dependent kinase activity is required for cells to enter the DNA synthetic (S) phase of their division cycle. Immature 32Dcl3 myeloid cells (32D) proliferating in the presence of interleukin-3 (IL-3) normally express cyclins D2 and D3, which assemble into binary holoenzyme complexes with their catalytic subunits, CDK4 and CDK6. When 32D cells are switched to medium containing granulocyte colony-stimulating factor (G-CSF ) instead of IL-3, D-type cyclins are degraded and, in the absence of their associated kinase activity, the cells arrest in the first gap phase (G1 ) of the cell cycle and differentiate to neutrophils. We derived 32D cells in which the expression of p19INK4d, a specific polypeptide inhibitor of CDK4 and CDK6, is regulated by the heavy metal-inducible sheep metallothionein promoter. Induction of p19INK4d in response to zinc prolonged cell survival in the absence of growth factor treatment. When maintained in medium containing both IL-3 and zinc, these cells lost cyclin D–dependent kinase activity, underwent G1 phase arrest, and acquired certain morphologic, antigenic, and functional properties of mononuclear phagocytes. Cells induced to express p19INK4d did not synthesize receptors for macrophage colony-stimulating factor (M-CSF/CSF-1) and reverted to an immature myeloid phenotype when shifted back into medium containing IL-3 alone. These cells exhibited accelerated differentiation to neutrophils in response to G-CSF but also gave rise to macrophage-like cells when maintained in medium containing both G-CSF and zinc. Therefore, the acquisition of macrophage properties in response to zinc treatment neither depended upon IL-3 nor upon G1 phase arrest per se and instead reflects some ability of p19INK4d, and presumably cyclin D–dependent kinases, to affect myeloid differentiation.

Features of Macrophage Differentiation Induced by p19INK4d, a Specific Inhibitor of Cyclin D–Dependent Kinases

AbstractThe mitogen-dependent induction of cyclin D–dependent kinase activity is required for cells to enter the DNA synthetic (S) phase of their division cycle. Immature 32Dcl3 myeloid cells (32D) proliferating in the presence of interleukin-3 (IL-3) normally express cyclins D2 and D3, which assemble into binary holoenzyme complexes with their catalytic subunits, CDK4 and CDK6. When 32D cells are switched to medium containing granulocyte colony-stimulating factor (G-CSF ) instead of IL-3, D-type cyclins are degraded and, in the absence of their associated kinase activity, the cells arrest in the first gap phase (G1 ) of the cell cycle and differentiate to neutrophils. We derived 32D cells in which the expression of p19INK4d, a specific polypeptide inhibitor of CDK4 and CDK6, is regulated by the heavy metal-inducible sheep metallothionein promoter. Induction of p19INK4d in response to zinc prolonged cell survival in the absence of growth factor treatment. When maintained in medium containing both IL-3 and zinc, these cells lost cyclin D–dependent kinase activity, underwent G1 phase arrest, and acquired certain morphologic, antigenic, and functional properties of mononuclear phagocytes. Cells induced to express p19INK4d did not synthesize receptors for macrophage colony-stimulating factor (M-CSF/CSF-1) and reverted to an immature myeloid phenotype when shifted back into medium containing IL-3 alone. These cells exhibited accelerated differentiation to neutrophils in response to G-CSF but also gave rise to macrophage-like cells when maintained in medium containing both G-CSF and zinc. Therefore, the acquisition of macrophage properties in response to zinc treatment neither depended upon IL-3 nor upon G1 phase arrest per se and instead reflects some ability of p19INK4d, and presumably cyclin D–dependent kinases, to affect myeloid differentiation.

Xid affects events leading to B cell cycle entry.

X-linked agammaglobulinemia patients and X-linked immunodeficient (xid) mice possess mutations in the Bruton's tyrosine kinase (Btk kinase) gene and display defects in B cell development and activation by sIg cross-linking. Btk is an early activation kinase in sIg-cross-linked B cells. xid does not ablate Btk protein kinase activity, and immediate signal transduction events, such as tyrosine phosphorylation, occur in sIg-activated xid B cells. These cells do not subsequently progress into cell division and have a high rate of apoptosis, which has been shown to correlate with an absence of sIg-mediated induction of the bcl-xL protein. To establish the point where Btk activity is critical for progression beyond immediate signaling, we examined early and late events in sIg-cross-linked xid B cells. Induction of proto-oncogenes and nuclear factors occurred normally in xid cells. However, induction of cyclins and increased GAPDH mRNA was not observed in xid cells. Degradation of the cyclin inhibitor p27Kip1 occurred normally in xid cells. After 24 h of culture with anti-mu, the remaining live, nonapoptotic xid cells were enlarged, viable, and primed for subsequent stimulation by LPS. Our data suggest that the Btk kinase is not essential for several G1 events and that the failure of sIg-activated xid B cells to enter cell cycle correlates with a defect of cyclin induction. Moreover, these data suggest that Btk is important not only for immediate events following B cell activation and control of apoptosis but also for subsequent events leading to cyclin activation.

P53 synthesis and phosphorylation in the aging diet-restricted rat following retinoic acid administration

Multiple doses of retinoic acid (RA) were administered intraperitoneally to three groups of male Fischer 344 rats over a 36 h period. The p53 isoforms from bone marrow nuclei in these three groups of rats were analyzed over time by two-dimensional polyacrylamide gel electrophoresis (PAGE) and fluorography for the incorporation of [ 35S]methionine (p53-synthesis) and [ 32P]phosphate (p53-phosphorylation). Two groups of rats, young (3.5 months) ad libitum (Y/AL) and old (28 months) ad libitum (O/AL), had free access to Purina rat chow; a third group of old (28 months) diet-restricted rats (O/DR) were maintained on a restricted caloric intake (60% of the AL diet) from 3 months of age. After 36 h of RA dosing, the PAGE patterns of p53 synthesis and phosphorylation in Y/AL and O/DR rats were very similar. In both groups, an increase in complexity was observed with labeling of additional isotypes possessing more acidic isoelectric values. In contrast, the O/AL animals showed a pattern of p53 isoform synthesis and phosphorylation that was considerably less complex and lacked the pronounced shift to more acidic forms following RA dosing. The p53 isoforms of O/AL rats as recognized by wild type (wt) Pab 246 antibody, were also much less dramatic in their increase to more acidic forms. Two-dimensional phospho-tryptic maps of Y/AL and O/DR rats were also very similar, both exhibiting two additional minor 32P-labeled fragments after RA dosing. The maps of O/AL rats did not show the two additional fragments following RA administration. After RA dosing, cyclin protein inhibitors (p16, p21, p27) revealed robust labeling with their respective antibodies in Y/AL and O/DR rats as analyzed by Western blotting. The O/AL animals showed marginally detectable antibody recognition of the cyclin inhibitors after RA dosing. Taken together, these data suggest that the biosynthesis and phosphorylation of p53 isoforms and the expression of cyclin dependent kinase inhibitor proteins is not significantly different between Y/AL and O/DR rats. Further, these results confirm and extend our previous observations that chronic diet-restriction attenuates the age related decline in the metabolic activity of nuclear protein products.

Analysis of phosphorylation of pRB and its regulatory proteins in breast cancer.

AIM: In order to study the role of retinoblastoma protein (pRB) in breast cancer, the phosphorylation of pRB and the expression of its related proteins-such as cyclin E, cyclin dependent...

P16/CDKN2 and CDK4 gene mutations in sporadic melanoma development and progression.

The p16/CDKN2(MTS1) gene encoding for the p16 inhibitor of cyclin D/CDK4 complexes is frequently mutated and deleted in a large fraction of melanoma cell lines, and p16 germline mutations have also been observed in familial melanomas. Moreover, a CDK4 gene mutation, responsible for a functional resistance of CDK4 kinase to p16 inhibitory activity, has been described to occur in some cases of familial melanoma. These data strongly support the idea that deregulation of the CDK4/cyclin D pathway, via CDKN2 or CDK4 mutations, is of biological significance in the development of melanoma. To shed light on the role of these alterations in the development and progression of sporadic melanoma, 12 primary melanomas and 9 corresponding metastases were analyzed for CDKN2 and CDK4 gene mutations. Of the 12 primary melanomas analyzed, 4 showed the presence of mutational inactivation of the p 16 protein and 2 carried silent mutations. No metastases showed the presence of CDKN2 mutations, indicating that mutations of this cyclin-dependent kinase inhibitor is not common in the progression of sporadic melanoma. On the other hand, the absence, in the metastases, of the CDKN2 mutation detected in the corresponding primary tumors suggests that 9p21 homozygous deletion may play a major role in the metastatic spreading of this type of tumor. None of the cases analyzed showed the presence of an Arg24Cys mutation, which functionally protects CDK4 from p16 inhibition. This indicates that CDK4 mutation plays a minor role in the development and progression of sporadic melanoma.

The ubiquitin-mediated proteolytic pathway as a therapeutic area.

Ubiquitin-mediated proteolysis is involved in the turnover of many short-lived regulatory proteins. This pathway leads to the covalent attachment of one or more multiubiquitin chains to target substrates which are then degraded by the 26S multicatalytic proteasome complex. Multiple classes of regulatory enzymes have been identified that mediate either ubiquitin conjugation or ubiquitin deconjugation from target substrates. Timed destruction of cellular regulators by the ubiquitin-proteasome pathway plays a critical role in ensuring normal cellular processes. This review provides multiple examples of key growth regulatory proteins whose levels are regulated by ubiquitin-mediated proteolysis. Pharmacological intervention which alters the half-lives of these cellular proteins may have wide therapeutic potential. Specifically, prevention of p53 ubiquitination (and subsequent degradation) in human papilloma virus positive tumors, and perhaps all tumors retaining wild-type p53 but lacking the retinoblastoma gene function, should lead to programmed cell death. Specific inhibitors of p27 and cyclin B ubiquitination are predicted to be potent antiproliferative agents. Inhibitors of IkappaB ubiquitination should prevent NFkappaB activation and may have utility in a variety of autoimmune and inflammatory conditions. Finally, we present a case for deubiquitination enzymes as novel, potential drug targets.