Cell-cycle Regulatory Proteins Cyclin D1 Research Articles

3200 – USING QUANTITATIVE PROTEOMICS TO IDENTIFY NEW THERAPEUTIC TARGETS IN MANTLE CELL LYMPHOMA

Mantle cell lymphoma (MCL) is a B cell non-Hodgkin's lymphoma that affects 4-6% of the population. Despite the relatively low incidence of MCL compared to other lymphomas, MCL only has a 5-year survival rate of 50%, making it one of the most deadly. While 90% of patients exhibit the t(11;14) translocation resulting in overexpression of the cell cycle regulatory protein CyclinD1 (CCND1), this alone is not enough for disease initiation. Patients must also have secondary mutations for disease manifestation; however, the number and variety of these mutations differ between every patient. This genetic complexity lends to the difficulty in disease treatment, and there are no standard therapies currently available. Although some therapies have shown promise for treatment in the early stages of the disease, all cases eventually stop responding, resulting in relapse. As such, it is imperative that new therapies be identified to treat this deadly disease. To this end, our lab performed quantitative proteomics on B cells from five healthy donors and tumors from different lymphoma patients including sixteen MCL, seven follicular lymphoma, and seven chronic lymphocytic lymphoma in order to identify changes at the proteomic level that are specific to MCL. This analysis has identified eight proteins that are specifically upregulated in MCL but are unchanged transcriptionally and would have gone unidentified by typical patient screening methods. Dysregulation of one of these proteins, HMGB3, has been shown to lend drug resistance in ovarian cancer and poor prognosis in neuroblastoma; however, has never been associated with MCL. Knockdown of HMGB3 in MCL cell lines results in reduced proliferation, increased apoptosis, and alterations to cell cycle, indicating it may prove to be a potential therapeutic target for MCL treatment. Mantle cell lymphoma (MCL) is a B cell non-Hodgkin's lymphoma that affects 4-6% of the population. Despite the relatively low incidence of MCL compared to other lymphomas, MCL only has a 5-year survival rate of 50%, making it one of the most deadly. While 90% of patients exhibit the t(11;14) translocation resulting in overexpression of the cell cycle regulatory protein CyclinD1 (CCND1), this alone is not enough for disease initiation. Patients must also have secondary mutations for disease manifestation; however, the number and variety of these mutations differ between every patient. This genetic complexity lends to the difficulty in disease treatment, and there are no standard therapies currently available. Although some therapies have shown promise for treatment in the early stages of the disease, all cases eventually stop responding, resulting in relapse. As such, it is imperative that new therapies be identified to treat this deadly disease. To this end, our lab performed quantitative proteomics on B cells from five healthy donors and tumors from different lymphoma patients including sixteen MCL, seven follicular lymphoma, and seven chronic lymphocytic lymphoma in order to identify changes at the proteomic level that are specific to MCL. This analysis has identified eight proteins that are specifically upregulated in MCL but are unchanged transcriptionally and would have gone unidentified by typical patient screening methods. Dysregulation of one of these proteins, HMGB3, has been shown to lend drug resistance in ovarian cancer and poor prognosis in neuroblastoma; however, has never been associated with MCL. Knockdown of HMGB3 in MCL cell lines results in reduced proliferation, increased apoptosis, and alterations to cell cycle, indicating it may prove to be a potential therapeutic target for MCL treatment.

MCM7 amplification and overexpression promote cell proliferation, colony formation and migration in esophageal squamous cell carcinoma by activating the AKT1/mTOR signaling pathway.

The roles and mechanisms of mini-chromosome maintenance complex component7 (MCM7) amplification and overexpression in esophageal carcinogenesis were investigated. By analyzing the TCGA datasets, we found that MCM7 was amplified in approximately 12% of esophageal squamous cell carcinomas (ESCCs), and in more than 4% of head and neck squamous cell carcinomas and stomach carcinomas. Overexpression of MCM7 was further verified in three independent GEO datasets of esophageal cancer. Knockdown of MCM7 using two siRNAs significantly inhibited cell proliferation, colony formation and migration of KYSE510 and EC9706 cells invitro. Noteworthy, we further found that silencing of MCM7 suppressed the phosphorylation of AKT1 and mTOR both in KYSE510 and EC9706 cells, and reduced the cell cycle regulatory proteins cyclinD1, cyclinE2 and CDK2. Taken together, our findings suggested that MCM7 promoted tumor cell proliferation, colony formation and migration of ESCC cells via activating AKT1/mTOR signaling pathway.

The EMSY Gene Collaborates with CCND1 in Non-Small Cell Lung Carcinogenesis.

Background: Lung cancer is the leading cause of cancer deaths. The main risk factor is smoking but the risk is also associated with various genetic and epigenetic components in addition to environmental factors. Increases in the gene copy numbers due to chromosomal amplifications constitute a common mechanism for oncogene activation. A gene-dense region on chromosome 11q13 which harbors four core regions that are frequently amplified, has been associated with various types of cancer. The important cell cycle regulatory protein cyclin D1 (CCND1) is an essential driver of the first core region of the Chr11q13 amplicon. Deregulation of CCND1 has been associated with different kinds of human malignancies including lung cancer. The EMSY (c11orf30) gene has been proposed as the possible driver of the fourth core of the 11q13 amplicon and its amplification has been associated with breast and ovarian cancers. There is no report in the literature investigating the EMSY gene in lung cancer.Methods: In this study, expression levels of the EMSY and CCND1 genes were investigated in 85 patients with non small cell lung cancer by Real Time PCR.Results: Expression of the EMSY and CCND1 genes were increased in 56 (65.8%) and 50 (58.8%) of the patients, respectively. Both genes showed a higher expression in the tumors when compared to normal tissues. A strong correlation was present between the expression rates of both genes (p<0.001). Patients with adenocarcinoma had higher expression levels of both genes (p=0.02).Conclusion: We conclude that EMSY and CCND1 work in collaboration and contribute to the pathogenesis of lung cancer.

CCND1 mutations increase protein stability and promote ibrutinib resistance in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation, which leads to deregulated expression of the cell cycle regulatory protein cyclin D1 (CCND1). Genomic studies of MCL have also identified recurrent mutations in the coding region of CCND1. However, the functional consequence of these mutations is not known. Here, we showed that, compared to wild type (WT), single E36K, Y44D or C47S CCND1 mutations increased CCND1 protein levels in MCL cell lines. Mechanistically, these mutations stabilized CCND1 protein through attenuation of threonine-286 phosphorylation, which is important for proteolysis through the ubiquitin-proteasome pathway. In addition, the mutant proteins preferentially localized to the nucleus. Interestingly, forced expression of WT or mutant CCND1 increased resistance of MCL cell lines to ibrutinib, an FDA-approved Bruton tyrosine kinase inhibitor for MCL treatment. The Y44D mutant sustained the resistance to ibrutinib even at supraphysiologic concentrations (5–10 μM). Furthermore, primary MCL tumors with CCND1 mutations also expressed stable CCND1 protein and were resistant to ibrutinib. These findings uncover a new mechanism that is critical for the regulation of CCND1 protein levels, and is directly relevant to primary ibrutinib resistance in MCL.

GSK3β/β-catenin signaling is correlated with the differentiation of glioma cells induced by wogonin

Malignant gliomas are the most common and most aggressive primary brain tumor, and for which differentiation therapy has emerged as a promising candidate strategy. In this study, we used in vitro and in vivo assays to examine the differentiation effects of wogonin, a major active constituent of Scutellaria baicalensis, on glioma C6 and U251 cells. We found that wogonin can suppress cell proliferation and induce G0/G1 arrest under a concentration-dependent manner. Wogonin also triggered significant reduction in the G1 cell-cycle regulatory proteins cyclin D1, cyclin-dependent kinase 2 and 4 along with overexpression of cell-cycle inhibitory proteins p27. Immunofluorescence and western blot analysis indicated that wogonin increased the expression of lineage-specific differentiation marker glial fibrillary acidic protein (GFAP). In mechanisms, we verified that wogonin significantly diminished the phosphorylated level of protein kinase B (AKT), and maintenance of low β-catenin expression level was dependent on glycogen synthase kinase 3β (GSK3β) activation at Ser9. Blocking GSK3β/β-catenin pathway was required for wogonin-induced proliferation inhibition and terminal differentiation by using canonical activator lithium chloride (LiCl) and inhibitor dickkopf-1 (Dkk1). Moreover, intravenous administration of wogonin delayed the growth of C6 glioma in the intracranial tumor model. These findings provide the evidence and mechanistic support for wogonin-based differentiation therapies for malignant glioblastoma. Furthermore, inhibition of GSK3β/β-catenin pathway may be a key and requisite factor in glioma differentiation.

A Synthetic Lethal RNA Interference Screen Identifies Components of Genotoxic Stress Response As Targets for Anti-CCND1 Therapy in Mantle Cell Lymphoma.

AbstractAbstract 2430 Background.Almost all cases of mantle cell lymphoma (MCL) harbor the t(11:14) chromosomal translocation resulting in overexpression of the cell cycle regulatory protein cyclin D1 (CCND1), which promotes cell proliferation and poor survival. Targeting CCND1 in vitro and clinically, however, is not sufficient to cause tumor cell death, suggesting that additional mechanisms compensate for MCL growth and survival. Unraveling these additional signals will help identify novel targets for rational combination therapies in MCL. Previously, we developed a novel functional genomics tool using an inducible RNA interference (RNAi) library, which can simultaneously assess the role of thousands of genes in cell viability in tumor cell lines. Here we apply the inducible RNAi screen to identify synthetic lethal interactions with CCND1 in MCL. The screen uncovered several components of the DNA damage response as potential new combination targets for anti-CCND1 therapy in mantle cell lymphoma. Methods.The RNAi library was previously constructed using a retroviral vector that inducibly expresses small-hairpin RNA (shRNA). Each shRNA vector contains a unique 60-mer bar code that can hybridize to a corresponding complementary sequence spotted on a custom Agilent oligonucleotide microarray. To perform a synthetic lethal RNAi screen for CCND1, we first established a stable MCL line (UPN-1) that express an inducible CCND1 or control shRNA, transduced these lines with the pooled shRNA library, selected for transduced cells with puromycin, and induced shRNA expression for eight days. Genomic DNA containing bar code sequences was then amplified by PCR, fluorescently labeled, and hybridized onto microarrays. Each screen was repeated four times to enable statistical analysis. Candidate shRNAs obtained from the screen were validated for synergistic killing of MCL cells when combined with CCND1 knockdown. We evaluated genotoxic stress response triggered by DNA damage following CCND1 inactivation in MCL lines by Western blots. DNA damage and repair were assessed by comet assays and immuno-fluorescent staining of DNA repair proteins including phospho-H2AX, RAD51 and 53BP1. Results.The RNAi screen uncovers multiple shRNAs targeting RIPK1, RIPK3, NEMO, and TAK1, which sensitize MCL cells to CCND1 inhibition. RIPK1, NEMO, and TAK1 have been shown to play an essential role in cells undergoing genotoxic stress by linking DNA damage-induced ATM activation and NF-kB activity. We demonstrated that silencing CCND1 in the MCL cell lines UPN-1, JEKO-1, Z138, and Granta-519 up-regulates RIPK1 mRNA and protein expression, in addition to increased phosphorylation of DNA damage response proteins such as ATM, CHEK1/2 and H2AX. We observed a two-fold increase of DNA damage levels in CCND1 knockdown cells as assessed by comet assays. We also detected cell cycle-independent increase of DNA double strand break (DSB) foci in CCND1 knockdown cells by staining with fluorescently labeled anti-phospho-H2AX antibody.Knockdown of RIPK1 in MCL lines (UPN-1 and JEKO-1) resulted in apoptotic cell death and these RIPK1 shRNA-transduced cells are hypersensitive to irradiation and DNA damaging agents, indicating RIPK1 plays a protective role against DNA damage-induced apoptosis. The survival role of RIPK1 in MCL cells may correlate with the DNA repair function as demonstrated by the inability of RIPK1 knockdown cells to efficiently resolve etoposide-induced DNA DSB foci over time. Furthermore, we also found that RIPK1 knockdown cells failed to down-regulate the G2/M cell cycle checkpoint protein CDC25B and to up-regulate ATM phosphorylation and Ku86 protein expression in response to genotoxic stress. Blocking these RIPK1-dependent responses could sensitize MCL cells to CCND1 knockdown-induced DNA damage. Similar analyses of the other hits from the RNAi screen are on going. Conclusions.There are few viable treatment options for mantle cell lymphoma. We have identified the receptor interacting protein kinase 1 (RIPK1), of the DNA damage response pathway, as a potential therapeutic target whose downregulation sensitizes MCL cells to anti-CCND1 treatment, possibly by promoting insurmountable genotoxic stress. Successful implementation of our functional genetic screens for genes that sensitize MCL cells to anti-CCND1 treatment could define novel targets suitable for effective combination therapies. Disclosures:No relevant conflicts of interest to declare.

The Effect of miR-338-3p on HBx Deletion-Mutant (HBx-d382) Mediated Liver-Cell Proliferation through CyclinD1 Regulation

ObjectiveHepatitis B Virus (HBV) DNA integration and HBV X (HBx) deletion mutation occurs in HBV-positive liver cancer patients, and C-terminal deletion in HBx gene mutants are highly associated with hepatocarcinogenesis. Our previous study found that the HBx-d382 deletion mutant (deleted at nt 382–400) can down-regulate miR-338-3p expression in HBx-expressing cells. The aim of the present study is to examine the role of miR-338-3p in the HBx-d382-mediated liver-cell proliferation.MethodsWe established HBx-expressing LO2 cells by Lipofectamine 2000 transfection. A miR-338-3p mimics or inhibitor was transfected into LO2/HBx-d382 and LO2/HBx cells using miR-NC as a control miRNA. In silico analysis of potential miR-338-3p targets revealed that miR-338-3p could target the cell cycle regulatory protein CyclinD1. To confirm that CyclinD1 is negatively regulated by miR-338-3p, we constructed luciferase reporters with wild-type and mutated CyclinD1-3′UTR target sites for miR-338-3p binding. We examined the CyclinD1 expression by real-time PCR and western blot, and proliferation activity by flow cytometric cell cycle analysis, Edu incorporation, and soft agar colony.ResultsHBx-d382 exhibited enhanced proliferation and CyclinD1 expression in LO2 cells. miR-338-3p expression inhibited cell proliferation in LO2/HBx-d382 cells (and LO2/HBx cells), and also negatively regulated CyclinD1 protein expression. Of the two putative miR-338-3p binding sites in the CyclinD1-3′UTR region, the effect of miR-338-3p on the second binding site (nt 2397–2403) was required for the inhibition.ConclusionmiR-338-3p can directly regulate CyclinD1 expression through binding to the CyclinD1-3′UTR region, mainly at nt 2397–2403. Down-regulation of miR-338-3p expression is required for liver cell proliferation in both LO2/HBx and LO2/HBx-d382 mutant cells, although the effect is more pronounced in LO2/HBx-d382 cells. Our study elucidated a novel mechanism, from a new miRNA-regulation perspective, underlying the propensity of HBx deletion mutants to induce hepatocarcinogenesis at a faster rate than HBx.

H-Ras mutation modulates the expression of major cell cycle regulatory proteins and disease prognosis in oral carcinoma

Activating mutations of the Ras is a moderately frequent event in oral carcinogenesis in Indian patients. Ras pathway has essential roles in regulation of various phases of the cell cycle, especially at G1 phase. Despite a large body of in vitro evidence, the multidimensional interaction between mutated Ras pathway and G1 cell cycle regulatory proteins in tumours in vivo is poorly determined. In the present study, DNA samples were screened for mutations in hot spot exons of B-Raf and hot spot codons 12, 13 and 61 of H-, K- and N-Ras by PCR-SSCP. Mutations were confirmed by direct sequencing. Expression of G1 cell cycle regulatory proteins—cyclin D1, CDK4, Rb, p53, p16 and p21 and proliferation marker PCNA was analysed immunohistochemically. The results revealed the absence of B-Raf mutations in oral carcinoma in spite of 12.5% of the samples showing H-Ras mutation. The H-Ras mutant cases showed significantly low cyclin D1 (P=0.027) and CDK4 (P=0.046) expression and overexpression of Rb (P=0.011) and p16 (P=0.026). H-Ras mutant carriers also had significantly high recurrence-free survival (P=0.033). In summary the present study demonstrated an epistatic interaction between H-Ras mutation and G1 cell cycle regulatory proteins in vivo. H-Ras mutation, thus, defines a molecular subtype of oral carcinoma with favourable outcome and unique biology.

The HSP90 inhibitor 17-AAG synergizes with doxorubicin and U0126 in anaplastic large cell lymphoma irrespective of ALK expression

Heat shock protein 90 (HSP90) chaperones and maintains the molecular integrity of a variety of signal transduction proteins, including the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein, a genetic abnormality that is frequently observed in anaplastic large cell lymphoma (ALCL) cells. Here we demonstrate that HSP90 is overexpressed in primary and cultured ALK-positive and ALK-negative ALCL cells, and we evaluate the potential role of the small molecule inhibitor of HSP90, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in treating ALCL. The antiproliferative effect of 17-AAG-cultured cells was determined by MTS assay. Apoptosis and cell-cycle arrest were determined by Annexin-V/propidium iodide and propidium iodide staining, respectively, and fluorescein-activated cell sorting analysis. Expression of HSP90 was evaluated by immunohistochemistry, and molecular changes were determined by Western blot. Treatment of cultured ALCL cells with 17-AAG induced cell-cycle arrest and apoptosis, irrespective of ALK expression. At the molecular level, 17-AAG induced degradation of ALK and Akt proteins, dephosphorylated extracellular signal-regulated kinase, and degraded the cell-cycle regulatory protein cyclin D1 and its cyclin-dependent kinases, CDK4 and CDK6, but had a differential effect on p27 and p53 proteins. Inhibition of extracellular signal-regulated kinase phosphorylation by the mitogen activated protein kinase inhibitor U0126 induced cell death in all ALCL cell lines, and sublethal concentration 17-AAG showed synergistic antiproliferative effects when combined with U0126 or doxorubicin. Our data demonstrate that targeting HSP90 function by 17-AAG may offer a novel therapeutic strategy for ALCL, either as single-agent activity or by combining 17-AAG with conventional or targeted therapeutic schemes.

Overexpression of cyclin D1 and cortactin is primarily independent of gene amplification in salivary gland adenoid cystic carcinoma

Adenoid cystic carcinoma (ACC) of the salivary glands exhibits persistent growth, invasion and metastasis. Chromosome 11q13 amplification is a frequent event associated with tumor progression in a number of carcinomas and is associated with poor prognosis. Two genes within the 11q13 amplicon that are overexpressed as a result of 11q13 amplification are the cell cycle regulatory protein cyclin D1 (CCND1) and cortactin (CTTN), a protein involved cell motility and invasion. To determine the expression and gene status of cyclin D1 and cortactin in ACC, we evaluated 39 ACC cases by immunohistochemistry (IHC) for cyclin D1 and cortactin expression. Amplification of CCND1 and CTTN was determined by fluorescent in situ hybridization (FISH). Cyclin D1 overexpression was present in 90% (35/39) and cortactin expression in 62% (24/39) of evaluated cases, although CCND1 and CTTN levels were elevated in only two cases (5%) as determined by FISH. Our results indicate that chromosome 11q13 amplification is uncommon in ACC, but that cyclin D1 and cortactin are frequently overexpressed and may therefore contribute to the growth and invasive potential of ACC.

Modulations of critical cell cycle regulatory events during chemoprevention of ultraviolet B-mediated responses by resveratrol in SKH-1 hairless mouse skin.

Multiple exposures to solar ultraviolet (UV) radiation cause critical damages that may lead to the development of several cutaneous disorders including skin cancer, the most frequently diagnosed malignancy in the USA. Therefore, efforts are needed to: (i) study the mechanism(s) of UV-mediated cutaneous damages, and (ii) design novel approaches for the management of skin cancer. 'Chemoprevention' via plant-based agents may be a useful approach for the management of neoplasia. Here, we evaluated the involvement of cell cycle regulatory molecules during resveratrol-mediated protection from multiple exposures of UVB (180 mJ/cm(2); on alternate days x 7 exposures) radiations in the SKH-1 hairless mouse skin. Resveratrol was topically applied on the skin of SKH-1 hairless mice at a dose of 10 micromol/mouse (in 0.2 ml acetone; 30 min prior to each UVB exposure). Studies were performed at 24 h following the last UVB exposure. Topical application of resveratrol resulted in significant decrease in UVB-induced bi-fold skin thickness, hyperplasia, and infiltration of leukocytes. The data from immunoblot and/or immunohistochemical analyses revealed that multiple exposure to UVB radiations causes significant upregulation in: (i) proliferating cell nuclear antigen (PCNA), a marker of cellular proliferation, and (ii) cyclin-dependent kinase (cdk)-2, -4 and -6, cyclin-D1, and cyclin-D2. Resveratrol treatment resulted in significant downregulation in UV-mediated increases in these critical cell cycle regulatory proteins. An interesting observation of this study was that resveratrol treatment resulted in a further stimulation of UVB-mediated increases in cyclin kinase inhibitor WAF1/p21 and tumor suppressor p53. Further, resveratrol was also found to cause significant decreases in UVB-mediated upregulation of: (i) the mitogen-activated protein kinase kinase, and (ii) the 42 kDa isotype of mitogen-activated protein kinase (MAPK). Thus, our data suggested that the antiproliferative effects of resveratrol might be mediated via modulation in the expression and function of cell cycle regulatory proteins cyclin-D1 and -D2, cdk-2, -4 and -6, and WAF1/p21. Our data further suggest that the modulation of cki-cyclin-cdk network by resveratrol may be associated with inhibition of the MAPK pathway. We suggest that resveratrol may be useful for the prevention of UVB-mediated cutaneous damages including skin cancer.

Ki-67 expression level, histological subtype, and the International Prognostic Index as outcome predictors in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is characterised by translocation t(11;14) (q13;q32) leading to rearrangement of bcl1/CCND1 and overexpression of the cell-cycle regulatory protein cyclin D1. We assessed the significance of the cell proliferation rate as an outcome predictor with the five components of the International Prognostic Index (IPI) and the histological subtypes of MCL. The hospital case records and histopathological material of 127 patients diagnosed with MCL in a single centre were reviewed. The cell proliferation rate was assessed by Ki-67 immunostaining and mitosis counting. Cox's multivariate regression model was used in multivariate survival analyses. The median follow-up time was 87 months. The mantle zone/nodular subtype of the common variant (19%) was associated with median survival of 70 months, the diffuse subtype of the common variant (64%) of 35 months, and the blastoid subtype (17%) of 11 months (P < 0.001). Patients with Ki-67 expression in > or = 26% (the upper tertile) of the lymphoma cells had median survival of only 13 months as compared with 45 months in the rest of the patients (P < 0.001). In a multivariate analysis high Ki-67 expression, Ann Arbor stage III-IV, and age over 60 yr had independent influence on survival, whereas serum lactate dehydrogenase level, the number of extranodal disease sites, and performance status did not. The IPI may not be an optimal tool for outcome prediction in MCL, and a better prognostic index may be obtained by including Ki-67 expression and possibly the histological subtype in the index.

Expression of the adhesion molecule CEACAM1 (CD66a, BGP, C-CAM) in breast cancer is associated with the expression of the tumor-suppressor genes Rb, Rb2, and p27.

The adhesion molecule CEACAM1 (CD66a, BGP, C-CAM) is not only involved in maintaining normal tissue architecture, but also acts as a tumor suppressor in several experimental systems where loss of CEACAM1 expression results in enhanced tumor-cell growth and tumorigenicity. In order to further analyze the role of CEACAM1 in the development of breast cancer, we performed Western-blot analysis and immunohistochemistry with highly specific monoclonal antibodies in a cohort of 68 mammary carcinomas which had also been analyzed for expression of cell-cycle regulatory proteins cyclin D1, cyclin E, p16, p21, p27, Rb, and Rb2, as well as for steroid hormone receptor status, Ki67, and HER2/neu immunoreactivity. High CEACAM1 protein expression as found using both methods correlated significantly with expression of the retinoblastoma proteins Rb (P=0.004 and 0.013) and Rb2/p130 (P=0.003 and 0.007). In addition, we found a weak association of CEACAM1 expression with p27 protein levels (P=0.087 and 0.039), but with none of the other analyzed parameters. These results indicate the possibility of a functional link between cell-adhesion molecules and cell-cycle regulation that might play an important role in the development of mammary carcinomas.

Expression of cell-cycle regulatory proteins cyclin D1, cyclin E, and their inhibitor p21 WAF1/CIP1 in gastric cancer.

The expression and prognostic role of cyclin D1, cyclin E, and p21 (WAF1/CIP1) were immunohistochemically investigated in 413 curatively resected gastric carcinomas. p21 was expressed in 65.4 per cent (n=270), cyclin D1 in 23.7 per cent (n=98), and cyclin E in 13.6 per cent ( n=56) of the tumours. The expression of p21, cyclin D1, and cyclin E was positively associated with the papillary or tubular type of the WHO classification, as well as with the intestinal type according to the Lauren classification. No significant correlation could be found between the expression of p21, cyclin D1 and cyclin E and the parameters pT category, lymph node involvement, and blood vessel and lymphatic vessel invasion. Concerning survival, no prognostic impact of p21, cyclin D1, and cyclin E expression could be verified, even when different subgroups of patients were analysed separately according to the pT and pN category as well as to the Lauren classification. The present data suggest that neither cyclin D1, cyclin E nor their inhibitor p21 can predict the survival of gastric cancer patients, nor is their immunohistochemical detection a suitable tool for identifying subgroups of patients who may be at higher risk.

Altered expression of the cell cycle regulatory protein cyclin D1 in the rat dentate gyrus after adrenalectomy-induced granular cell loss

The loss of dentate gyrus (DG) granular cells after removal of the rat adrenal glands (ADX) is mediated by a process that is apoptotic in nature. The present study was initiated to compare changes in the immunocytochemical distribution of the cell-cycle regulatory protein cyclin D1, which has been implicated in apoptosis, with the loss of DG granular cells after ADX. Our data indicate that cyclin D1-immunoreactivity (cyclin D1-ir) is enhanced in the rat dentate gyrus after adrenalectomy. The enhanced cyclin D1-ir shows a close relationship, both in time and space, with granular cell loss in the rat dentate gyrus that occurs after adrenalectomy. However, the enhanced cyclin D1-immunoreactivity was present in microglia and radial glia rather than in the dentate gyrus granular cells. This suggests that cyclin D1 is not directly involved in apoptosis of granular cells in the rat dentate gyrus after adrenalectomy.