Biological implications of the inhibition of survivin by RNA interference in human androgen-independent prostate carcinoma with highly metastatic potential / 中华病理学杂志

<p><b>OBJECTIVE</b>To determine the expression level of survivin in androgen-independent prostate carcinoma, and to investigate the biological role of survivin in invasion and metastasis of androgen-independent prostate carcinoma.</p><p><b>METHODS</b>Highly metastatic prostatic cancer cell line PC-3M-1E8 was stably transfected with pSilencer plasmid targeting survivin expression by RNA interference. The biological effects were observed, including anchorage-independent growth, in vitro invasion by soft agar colony formation and Boyden chamber assay, and also in vivo tumorigenesis in nude mice. Cell cycle and apoptosis indices were evaluated by flow cytometry and Western blot analysis of bioactive fragments of caspase 3.</p><p><b>RESULTS</b>The expression of survivin in transfected PC-3M-1E8 cells was markedly depressed at both mRNA and protein levels (about 78% to 80%) as compared with control. The growth of tumor cells was retarded by anchorage-independent growth assay. The survivin transfectants formed smaller and fewer colonies (14.33 +/- 3.51) than the negative (52.33 +/- 6.81) and blank controls (54.00 +/- 6.00). Inhibition of survivin expression was correlated with enhanced apoptosis of tumor cells (percentages of apoptotic cells of the negative control, blank control and experimental groups were 5.88 +/- 0.99, 6.97 +/- 1.60, 16.40 +/- 1.95 respectively), along with an increased expression of activated caspase 3, and cell cycle inhibition at G(0)/G(1) phase (the relative number of cells at G(0)/G(1) phase were 43.65 +/- 3.44, 43.59 +/- 1.83 and 52.71 +/- 1.10, respectively). In addition, multinucleated giant cells were observed along with a marked inhibition of invasion as reflected by fewer penetrating cells by Boyden chamber assay (46.07 +/- 9.97, 47.87 +/- 9.58 and 38.67 +/- 6.59, respectively).</p><p><b>CONCLUSIONS</b>Survivin expression is high in androgen-independent prostate cancer cells and likely may be related to the apoptosis, growth and invasion of the tumor cells. Targeting the survivin pathway by RNA interference appears to be a promising approach for clinical treatment of androgen-independent prostate cancer.</p>

Monoclonal antibodies against human tumor metastasis suppressor gene-1 TMSG-1: preparation, characterization and application / 中华病理学杂志

<p><b>OBJECTIVE</b>In order to clarify the exact molecular weight of tumor metastasis suppressor gene-1 (TMSG-1) protein and its cellular localization, a monoclonal antibody against TMSG-1 was prepared, characterized and applied to evaluate the metastatic potential of human tumors.</p><p><b>METHODS</b>A dominant epitope-TMSG-1(15)-derived from TMSG-1 was synthesized based on Fmoc method, and the hapten was conjugated to Imject Maleimide activated mcKLH as a carrier protein. The antigen preparation was used to immunize BAL B/C mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, which were further characterized by western blotting and immunohistochemical staining.</p><p><b>RESULTS</b>One hybridoma cell line secreting anti-TMSG-1 antibody, designated as C8, was eventually established after primary ELISA screening, followed by rapid limited dilution procedure. It was confirmed that C8 was of IgM isotype. Result of competitive inhibition assay showed that the antibody was TMSG-1 specific. Using this antibody, an expected protein band of about 45,000 (relative molecular mass) was detected in the non-metastatic variants PC(3)-2B4 and PG-LH7 cells by Western blotting, but not in the isogenetic metastatic variants of PC3-1E8 and PG-BE1 cells. Immunohistochemistry using C8 showed a positive staining of cell membrane and cytoplasm of 2B4 and LH7 cells, whereas 1E8 and BE1 cells were non-reactive. Immunostaining using C8 of paraffin sections of 52 breast carcinomas and 41 colon cancers demonstrated a strong positivity in non-metastatic tumors, but none to weakly reactive in metastatic tumors (P < 0.05).</p><p><b>CONCLUSION</b>C8 monoclonal antibody against the synthetic peptide is TMSG-1 specific and is effective for Western blot and immunohistochemistry assays to detect TMSG-1 expression in cancer cells. TMSG-1 protein is about 45 000 (relative molecular mass) at cell membrane and cytoplasm of tumor cells. Expression of TMSG-1 protein correlates well, inversely with the tumor metastatic potential.</p>

Monoclonal antibody against G3BP: preparation, characterization and its application in analysis of human tumors / 中华病理学杂志

<p><b>OBJECTIVE</b>To better understand the molecular mechanism of tumorigenesis and progression, the monoclonal antibody against G3BP (Ras-GAP SH3 binding protein), which serves as an important downstream effector of Ras signaling, was prepared, characterized and utilized in analysis of various human tumors.</p><p><b>METHODS</b>By using the prokaryotic expression vector pGEX-5X1, GST-G3BP fusion protein was expressed in E. coli BL21 under induction of IPTG. Purified GST-G3BP fusion protein was used to immunize BALB/c mice. The monoclonal antibody against G3BP was produced through conventional hybridoma method and characterized by ELISA, Western blot and immunohistochemical staining.</p><p><b>RESULTS</b>A hybridoma cell line secreting anti-G3BP IgG1 subtype antibody was obtained. Western blot and competitive inhibition assay showed that the antibody was G3BP-specific. Immunohistochemical staining demonstrated that G3BP was over-expressed in formalin-fixed and paraffin-embedded tissues of some human tumors, such as lung cancer, colon cancer, gastric cancer and breast cancer. In breast cancer specimens, the degree of G3BP expression correlated positively with the presence of lymph node metastasis and c-erbB2 expression.</p><p><b>CONCLUSIONS</b>The G3BP-specific monoclonal antibody derived from recombination protein can be used in ELISA, Western blot and immunohistochemical assay. It may provide an important tool in analysis of G3BP in in vitro and in vivo experiments. Besides, G3BP may serve as another prognostic marker for breast cancer.</p>

Thymosin beta10 expression and actin filament organization in tumor cell lines with different metastatic potential / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the expression of thymosin beta10 (Tbeta10) and related changes of actin filament organization in human tumor cell lines with different metastatic potential.</p><p><b>METHODS</b>Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the expression of Tbeta10 mRNA detected by northern-blot and its peptide by immunohistochemical staining. The filamentous actin (F-actin) was stained with TRITC-phalloidin to detect changes in actin organization.</p><p><b>RESULTS</b>In comparison with the non and/or weakly metastatic counterparts, Tbeta10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. TRITC-phalloidin staining revealed less actin bundles and a fuzzy network of shorter filaments in the highly metastatic tumor cells, while in the non and/or weakly metastatic cancer cell lines, there were thick and orderly arranged actin filaments.</p><p><b>CONCLUSIONS</b>Tbeta10 levels correlate positively with the metastatic phenotype in human tumors currently examined. The increased metastatic potential of tumor cells is accompanied by the loss of F-actin and poorly organized actin skeleton. There is a consistent correlation between the elevated Tbeta10 expression and the disrupted actin skeleton.</p>

Differential thymosin beta 10 expression levels and actin filament organization in tumor cell lines with different metastatic potential / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>To investigate the differential expression levels of thymosin beta 10 (T beta 10) and the corresponding changes of actin filament organization in human tumor cell lines with different metastatic potential.</p><p><b>METHODS</b>Four groups of nine human tumor cell lines with different metastatic potential were analyzed for the amount of T beta 10 mRNAs by Northern blot and for their peptide expression levels by immunohistochemistry. The filamentous actin (F-actin) was observed by staining of TRITC-phalloidin to detect changes in actin organization.</p><p><b>RESULTS</b>In comparison with non-/weakly metastatic counterparts, T beta 10 was upregulated in highly metastatic human lung cancer, malignant melanoma and breast cancer cell lines. Staining of TRITC-phalloidin revealed less actin bundles, a fuzzy network of shorter filaments and some F-actin aggregates in the highly metastatic tumor cells. Meanwhile, the actin filaments were robust and orderly arranged in the non-/weakly metastatic cancer cell lines.</p><p><b>CONCLUSION</b>T beta 10 levels correlate positively with the metastatic capacity in human tumors currently examined. The increasing metastatic potential of tumor cells is accompanied by a loss of F-actin, poorly arranged actin skeleton organizations and presence of F-actin aggregates. There is a consistent correlation between the elevated T beta 10 expression and the disrupted actin skeleton.</p>