Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^

Invasive phenotype of MCF10A cells overexpressing c-Ha-ras and c-erbB-2 oncogenes

Infection with erbB-2 (E) of Ha-ras (H) oncogene-transfected cells has been previously shown to cooperatively induce anchorage-independent growth of the MCF10A human mammary epithelial cell line in vitro, but not to induce nude mouse tumorigenicity. Here we show that oncogene-transformed MCF10A are able to halt in the lungs of nude mice, a sign of organ colonization potential. We have therefore studied the transformants for in vitro migratory and invasive properties known to correlate with the metastatic potential of human mammary carcinoma cells in nude mice. MCF10A transfected with Ha-ras, infected with a recombinant retroviral vector containing the human c-erB-2 proto-oncogene (MCF10A-HE cells), show a higher invasive index than either the single transfectant (MCF10A-H) or MCF10A-erB-2(MCF10A-E) cells in the Boyden chamber chemotaxis and chemoinvasion assays. The MCF10A-HE cells also adopted an invasive stellate growth pattern when plated or embedded in Matrigel, in contrast to the spherical colonies formed by the single transformants MCF10A-H, MCF10A-E, and the parental cells. Dot-blot analysis of gelatinase A and TIMP-2 mRNA levels revealed increasing gelatinase A mRNA levels (HE > E > H > MCF10A) and reduced TIMP-2 expression in both single and double transformants. Furthermore, MCF10A-HE cells show more MMP-2 activity than parental MCF10A cells or the single transformants. CD44 analysis revealed differential isoform banding for the MCF10A-HE cells compared to parental cells, MCF10A-H and MCF10A-E, accompanied by increased binding of hyaluronan by the double transformants. Our results indicate that erB-2 and Ha-ras co-expression can induce a more aggressive phenotype in vitro, representative of the malignancy of mammary carcinomas.

Chk1 Suppresses a Caspase-2 Apoptotic Response to DNA Damage that Bypasses p53, Bcl-2, and Caspase-3

Evasion of DNA damage-induced cell death, via mutation of the p53 tumor suppressor or overexpression of prosurvival Bcl-2 family proteins, is a key step toward malignant transformation and therapeutic resistance. We report that depletion or acute inhibition of checkpoint kinase 1 (Chk1) is sufficient to restore ?-radiation-induced apoptosis in p53 mutant zebrafish embryos. Surprisingly, caspase-3 is not activated prior to DNA fragmentation, in contrast to classical intrinsic or extrinsic apoptosis. Rather, an alternative apoptotic program is engaged that cell autonomously requires atm (ataxia telangiectasia mutated), atr (ATM and Rad3-related) and caspase-2, and is not affected by p53 loss or overexpression of bcl-2/xl. Similarly, Chk1 inhibitor-treated human tumor cells hyperactivate ATM, ATR, and caspase-2 after ?-radiation and trigger a caspase-2-dependent apoptotic program that bypasses p53 deficiency and excess Bcl-2. The evolutionarily conserved "Chk1-suppressed" pathway defines a novel apoptotic process, whose responsiveness to Chk1 inhibitors and insensitivity to p53 and BCL2 alterations have important implications for cancer therapy. © 2008 Elsevier Inc. All rights reserved.

Microtubule disrupting N-phenyl-N’-(2-chloroethyl) ureas display anticancer activity on cell adhesion, P-glycoprotein and BCL-2-mediated drug resistance.

Objective: Our research program has focused on the development of promising, soft alkylating N-phenyl-N’-(2-chloroethyl)urea (CEU) compounds which acylate the glutamic acid-198 of β-tubulin, near the binding site of colchicum alkaloids. CEUs inhibit the motility of cancerous cells in vitro and, interestingly, exhibit antiangiogenic and anticancer activity in vivo. Mitotic arrest induced by microtubule-interfering agents such as CEUs remains the major mechanism of their anticancer activity, leading to apoptosis. However, we recently demonstrated that microtubule disruption by CEUs and other common antimicrotubule agents greatly alters the integrity and organization of microtubule-associated structures, the focal adhesion contact, thereby initiating anoikis, an apoptosis-like cell death mechanism caused by the loss of cell contact with the extracellular matrix. Methods: To ascertain the activated signaling pathway profile of CEUs, flow cytometry, Western blot, immunohistochemistry and transfection experiments were performed. Wound-healing and chick embryo assays were carried out to evaluate the antiangiogenic potency of CEUs. Results: CEU-induced apoptosis involved early cell cycle arrest in G2/M and increased level of CDK1/cycline B proteins. These signaling events were followed by the specific activation of the intrinsic apoptosis pathway, involving loss of mitochondrial membrane potential (Δψm) and ROS production, cytochrome c release from mitochondria, caspase activation, AIF nuclear translocation, PARP cleavage and nuclear fragmentation. CEUs maintained their efficacy on cells plated on pro-survival extracellular matrices or exhibiting overexpression of P-glycoprotein or the anti-apoptotic protein Bcl-2. Conclusion: Our results suggest that CEUs represent a promising new class of antimicrotubule, antiangiogenic and pro-anoikis agents.

Analysis of Ki-67 and Bcl-2 protein expression in normal colorectal mucosa of women with breast cancer

The aim of this study was to evaluate Ki-67 and Bcl-2 protein expression in the normal colorectal mucosa adjacent to adenomatous polyps in women with breast cancer. A cross-sectional, controlled study was conducted in 35 women with and without breast cancer who had adenomatous colorectal polyps. The patients were divided into two groups: Group A (a control group of women without breast cancer, n = 18) and Group B (a study group of women with breast cancer, n = 17). A sample of normal colonic mucosa was collected at a distance of 5 cm from the polypoid lesion to evaluate immunchistochemical expression of the Ki-67 and Bcl-2 proteins. Student`s t-test and the chi-square test were used to analyse Ki-67 and Bcl-2 expression, respectively. Statistical significance was established at p < 0.05. The mean percentage of Ki-67-stained nuclei in Groups A and B was 25.12 +/- 2.08 and 41.50 +/- 1.85, respectively (p < 0.001), whereas the percentage of cases with cells expressing Bcl-2 in Groups A and B was 17.6% and 82.4%, respectively (p < 0.003). In the present study, greater proliferative activity and greater expression of the antiapoptotic protein Bcl-2 was found in the normal colorectal mucosa of women with breast cancer. (C) 2009 Elsevier Ltd. All rights reserved.

Expressão gênica do receptor estrogênico-a, bcl-2 e c-myc em fibroadenomas e no tecido mamário normal circunjacente

Resumo não disponível.

Comparative analysis of the expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma

Several epidermiologic studies have shown the malignant transformation potential of oral lichen planus; however, this potential is subject of much controversy. To evaluate the expression of proteins related to the cell proliferation and apoptosis processes in oral lichen planus, we compared oral lichen planus with oral squamous cell carcinoma. Twenty-four cases of each lesion were submitted according to streptavidin-biotin technique to evaluate the immunohistochemical expression of proliferating cell nuclear antigen, p53, bax, and bcl-2 proteins. X 2 test showed no statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma (P > .05). However, the expression of proliferating cell nuclear antigen was significantly lower in oral lichen planus than in oral squamous cell carcinoma (P < .05). No statistically significant differences between the expression of p53, bax, and bcl-2 in oral lichen planus and oral squamous cell carcinoma were observed, which may be an evidence of the potential of malignant transformation of oral lichen planus. (C) 2009 Elsevier B.V. All rights reserved.

Serum Metalloproteinases 2 and 9 as Predictors of Gait Status, Pressure Ulcer and Mortality after Hip Fracture

Introduction: The aim of this study is to evaluate the serum activity of metalloproteinases (MMPs) -2 and -9 as predictors of pressure ulcer (PU), gait status and mortality 6 months after hip fracture. Methods: Eighty-seven patients over the age of 65 admitted to the orthopedic unit from January to December 2010 with hip fracture were prospectively evaluated. Upon admission, patient demographic information, including age, gender and concomitant diseases, was recorded. Blood samples were taken for analysis of MMP -2 and -9 activity by gel zymography and for biochemical examination within the first 72 hours of the patient's admission, after clinical stabilization. The fracture pattern (neck, trochanteric or subtrochanteric), time from admission to surgery, surgery duration and length of hospital stay were also recorded. Results: Two patients were excluded due to the presence of pathological fractures (related to cancer), and three patients were excluded due to the presence of PU before admission. Eighty-two patients, with a mean age of 80.4 ± 7.3 years, were included in the analysis. Among these patients, 75.6% were female, 59.8% had PU, and 13.4% died 6 months after hip fracture. All patients underwent hip fracture repair. In a univariate analysis, there were no differences in serum MMP activity between hip fracture patients with or without PU. In addition, the multiple logistic regression analysis models, which were adjusted by age, gender, length of hospital stay and C-reactive protein, showed that the pro-MMP-9 complexed with neutrophil gelatinase-associated lipocalin form (130 kDa) was associated with gait status recovery 6 months after hip fracture. Conclusions: In conclusion, serum pro-MMP-9 is a predictor of gait status recovery 6 months after hip fracture. © 2013 Gumieiro et al.

Detection of the tumour suppressor gene TP53 and expression of p53, Bcl-2 and p63 proteins in canine transmissible venereal tumour

Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.

Mutation and genomic amplification of the PIK3CA proto-oncogene in pituitary adenomas

The tumorigenesis of pituitary adenomas is poorly understood. Mutations of the PIK3CA proto-oncogene, which encodes the p110-α catalytic subunit of PI3K, have been reported in various types of human cancers regarding the role of the gene in cell proliferation and survival through activation of the PI3K/Akt signaling pathway. Only one Chinese study described somatic mutations and amplification of the PIK3CA gene in a large series of pituitary adenomas. The aim of the present study was to determine genetic alterations of PIK3CA in a second series that consisted of 33 pituitary adenomas of different subtypes diagnosed by immunohistochemistry: 6 adrenocorticotropic hormone-secreting microadenomas, 5 growth hormone-secreting macroadenomas, 7 prolactin-secreting macroadenomas, and 15 nonfunctioning macroadenomas. Direct sequencing of exons 9 and 20 assessed by qPCR was employed to investigate the presence of mutations and genomic amplification defined as a copy number ≥4. Previously identified PIK3CA mutations (exon 20) were detected in four cases (12.1%). Interestingly, the Chinese study reported mutations only in invasive tumors, while we found a PIK3CA mutation in one noninvasive corticotroph microadenoma. PIK3CA amplification was observed in 21.2% (7/33) of the cases. This study demonstrates the presence of somatic mutations and amplifications of the PIK3CA gene in a second series of pituitary adenomas, corroborating the previously described involvement of the PI3K/Akt signaling pathway in the tumorigenic process of this gland.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.