Expression profiling of cell cycle regulatory proteins in oropharyngeal carcinomas using tissue microarrays.

AIM: The aim of this study was to investigate the expressions of cell cycle regulatory proteins such as p53, p16, p21, and Rb in squamous cell carcinoma of the oropharynx and their relation to histological differentiation, staging of disease, and prognosis. PATIENTS AND METHODS: Paraffin blocks from 21 primary tumors were obtained from archives of the Department of Pathology, Paulista Medical School, Federal University of Sao Paulo, UNIFESP/EPM. Immunohistochemistry was used to detect the expression of p53, p16, p21, and Rb by means of tissue microarrays. RESULTS: Expression of p53, p21, p16 and Rb was not correlated with the stage of disease, histopathological grading or recurrence in squamous cell carcinoma of the oropharynx. CONCLUSION: Taken together, our results suggest that p53, p16, p21 and Rb are not reliable biomarkers for prognosis of the tumor severity or recurrence in squamous cell carcinoma of the oropharynx as depicted by tissue microarrays and immunohistochemistry.

Amifostine Increases FAS and Caspase-3 Expression in Colonic Tissue of Irradiated Mice.

BACKGROUND/AIM: The aim of this study was to evaluate the expression of FASL, FAS and FADD and caspase-3 in oesophagus, stomach and colonic tissues of mice irradiated in vivo by immunohistochemistry. MATERIALS AND METHODS: A total of 48 adult male C57BL mice were distributed into four groups: Ami(-)/Rad(-): Mice received 0.5 ml of 0.9% physiological saline solution (PPS) intraperitioneally (i.p.); Ami(+)/Rad(-): mice received amifostine (400mg/kg i.p.) freshly dissolved in double-distilled water; Ami(-)/Rad(+): mice received 0.5 ml of PSS i.p. 30 min before a single whole-body radiation dose of 7 Gy; Ami(+)/Rad(+): mice received 0.5 ml of an aqueous solution of 400 mg/kg amifostine i.p.30 min prior to irradiation. All groups were assigned into subgroups sacrificed at 0.5 h, 1 h, 2 h and 4 h after irradiation. RESULTS: In oesophagus and stomach tissues, we did not observe any difference between Ami(-)/ad(-), Ami(+)/Rad(-), Ami(-)/Rad(+) and Ami(+)/Rad(+) groups in the expression of FASL, FAS and FADD. The colonic tissue was the only to exhibit any difference in the expression of FAS and caspase-3 protein in the Ami(-)/Rad(+)group at 1 and 2 h. Amifostine increased FAS and caspase-3 immunoexpression when compared to the control. Immunoexpression for FASL and FADD was not remarkably different in colonic tissue. CONCLUSION: Taken together, our results demonstrate that amifostine increases FAS and caspase-3 expression in colonic tissue of irradiated mice.

The extrinsic apoptotic signaling pathway in gastric adenocarcinomas assessed by tissue microarray.

The purpose of this investigation was to analyze the immunoexpression of FasL, Fas, FADD, cleaved caspase 8, and cleaved caspase 3 in gastric cancer. Formalin-fixed and paraffin-embedded gastric adenocarcinoma tissues from 87 patients, including adjacent normal tissues, were included on tissue microarray by immunohistochemistry. The tumor and the adjacent normal tissues were positive for FasL in 66.7% and 90.6%, for Fas in 52.8% and 52.4%, for FADD in 67.4% and 82.3%, for cleaved caspase 8 in 27.9% and 37.7%, and for cleaved caspase 3 in 33.7% and 8.3%, respectively. FasL and the FADD from tumor were statistically different in relation to the histological type. Cleaved caspase 8 was statistically different in relation to clinical stage (p=0.031). The FADD from normal tissue was statistically different in relation to age (p=0.039), sex (p=0.055), clinical stage (p=0.019), and Fas was different in relation to tumor size (p=0.012). In the tumor, we observed a correlation between FasL and Fas, FasL and FADD, and FasL and cleaved caspase 3. In the adjacent normal tissue, a correlation was observed between FasL and Fas, FasL and FADD. There was no association of another marker with sex, age, clinical stage, and survival. Our results suggest that these proteins mediate the early extrinsic apoptotic pathway in gastric cancer and adjacent normal mucosa. FasL protein binds to Fas protein and subsequently binds to death receptor FADD signaling activation of the extrinsic apoptotic pathway. In this phase, there was inhibition of caspase 8 and, consequently, decreased apoptosis.

Expression of p27, p21(WAF/Cip1), and p16(INK4a) in normal oral epithelium, oral squamous papilloma, and oral squamous cell carcinoma.

AIM: To characterize the immunohistochemical expression of p27, p21(WAF/Cip1) and p16(INK4a) in normal oral epithelium, oral squamous papilloma and oral squamous cell carcinoma. MATERIALS AND METHODS: Immunohistochemical staining for p27, p21(WAF/Cip1) and p16(INK4a) was evaluated in 32 samples of normal oral squamous epithelium, 30 samples of oral squamous papilloma, and 34 samples of oral squamous cell carcinoma. RESULTS: Statistically significant differences (p<0.05) were found in p27 expression when comparing ordinary mucosa and oral squamous papilloma with the oral squamous cell carcinoma samples. Regarding p21(WAF/Cip1) expression, no statistically significant differences (p>0.05) were noticed. In the same way, no significant statistically differences (p>0.05) were observed for p16(INK4a) among groups. CONCLUSION: Taken together, these findings indicate that p27 is closely involved in malignant transformation of oral mucosa cells, and may be a reliable biomarker for this purpose.

The intrinsic apoptotic signaling pathway in gastric adenocarcinomas of Brazilian patients: Immunoexpression of the Bcl-2 family (Bcl-2, Bcl-x, Bak, Bax, Bad) determined by tissue microarray analysis.

This study was undertaken to investigate the immunoexpression of Bcl-2 family proteins (Bcl-2, Bcl-x, Bax, Bak and Bad) and to evaluate the correlation between the immunoexpression of these proteins and that of cleaved caspase 3, Ki-67 and p53. A tissue microarray (TMA) paraffin block was constructed using gastric carcinoma tissue (test group) and adjacent normal gastric mucosa (control group) from 87 patients who had not previously undergone radiotherapy or chemotherapy. Sections from the TMA block (4 µm) were subjected to immunohistochemistry for Bcl-2, Bcl-x, Bax, Bak, Bad, p53, Ki-67 and cleaved caspase-3. The slides were evaluated by the semi-quantitative method, and the scores obtained (intensity vs. percentage of staining) were correlated with one another and with the apoptotic index, cellular proliferation and data regarding patient survival. The studied proteins were present in the tumor tissue and in the normal gastric mucosa, but at different intensities and with differences in the number of positive cells. There was an association between tumor size and p53 expression, and intestinal type adenocarcinoma was positively correlated with the expression of Bax, Bad and Ki-67. The immunoexpression of Bcl-x, Bak, Bad, p53 and Ki-67 showed statistically significant differences between the tumor tissue and the adjacent normal gastric mucosa. There was an association between the expression of Bax, Bak and Bad in the normal gastric mucosa. No correlation between patient survival and the expression of these proteins was observed. Overexpression of the Bcl-x protein in the adenocarcinomas and the difference in Bcl-x expression between the test and the control group may be related to the anti-apoptotic effect of this protein. The reduced expression of Bak and Bad and the increased expression of p53 and Ki-67 in the adenocarcinomas demonstrate the imbalance between apoptosis and cellular proliferation, which results in uncontrolled tumor cell proliferation.

Receptor-induced dilatation in the systemic and intrarenal adaptation to pregnancy in rats.

Normal pregnancy is associated with systemic and intrarenal vasodilatation resulting in an increased glomerular filtration rate. This adaptive response occurs in spite of elevated circulating levels of angiotensin II (Ang II). In the present study, we evaluated the potential mechanisms responsible for this adaptation. The reactivity of the mesangial cells (MCs) cultured from 14-day-pregnant rats to Ang II was measured through changes in the intracellular calcium concentration ([Cai]). The expression levels of inducible nitric oxide synthase (iNOS), the Ang II-induced vasodilatation receptor AT2, and the relaxin (LGR7) receptor were evaluated in cultured MCs and in the aorta, renal artery and kidney cortex by real time-PCR. The intrarenal distribution of LGR7 was further analyzed by immunohistochemistry. The MCs displayed a relative insensitivity to Ang II, which was paralleled by an impressive increase in the expression level of iNOS, AT2 and LGR7. These results suggest that the MCs also adapt to the pregnancy, thereby contributing to the maintenance of the glomerular surface area even in the presence of high levels of Ang II. The mRNA expression levels of AT2 and LGR7 also increased in the aorta, renal artery and kidney of the pregnant animals, whereas the expression of the AT1 did not significantly change. This further suggests a role of these vasodilatation-induced receptors in the systemic and intrarenal adaptation during pregnancy. LGR7 was localized in the glomeruli and on the apical membrane of the tubular cells, with stronger labeling in the kidneys of pregnant rats. These results suggest a role of iNOS, AT2, and LGR7 in the systemic vasodilatation and intrarenal adaptation to pregnancy and also suggest a pivotal role for relaxin in the tubular function during gestation.