Skeletal Muscle Phenotypically Converts and Selectively Inhibits Metastatic Cells in Mice

Skeletal muscle is rarely a site of malignant metastasis; the molecular and cellular basis for this rarity is not understood. We report that myogenic cells exert pronounced effects upon co-culture with metastatic melanoma (B16-F10) or carcinoma (LLC1) cells including conversion to the myogenic lineage in vitro and in vivo, as well as inhibition of melanin production in melanoma cells coupled with cytotoxic and cytostatic effects. No effect is seen with non-tumorigenic cells. Tumor suppression assays reveal that the muscle-mediated tumor suppressor effects do not generate resistant clones but function through the down-regulation of the transcription factor MiTF, a master regulator of melanocyte development and a melanoma oncogene. Our findings point to skeletal muscle as a source of therapeutic agents in the treatment of metastatic cancers

Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

111-118<span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension in to subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted sc in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, <span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (2 1 %) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as <span style="font-size: 15.0pt;mso-bidi-font-size:8.0pt;font-family:" times="" new="" roman","serif""="">B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation. </span

p53-mediated induction of Cox-2 counteracts p53- or genotoxic stress-induced apoptosis

The identification of transcriptional targets of the tumor suppressor p53 is crucial in understanding mechanisms by which it affects cellular outcomes. Through expression array analysis, we identified cyclooxygenase 2 (Cox-2), whose expression was inducible by wild-type p53 and DNA damage. We also found that p53-induced Cox-2 expression results from p53-mediated activation of the Ras/Raf/MAPK cascade, as demonstrated by suppression of Cox-2 induction in response to p53 by dominant-negative Ras or Raf1 mutants. Furthermore, heparin-binding epidermal growth factor-like growth factor (HB- EGF), a p53 downstream target gene, induced Cox-2 expression, implying that Cox-2 is an ultimate effector in the p53→HB-EGF→Ras/Raf/MAPK→Cox-2 pathway. p53-induced apoptosis was enhanced greatly in Cox-2 knock-out cells as compared with wild-type cells, suggesting that Cox-2 has an abrogating effect on p53-induced apoptosis. Also, a selective Cox-2 inhibitor, NS-398, significantly enhanced genotoxic stress-induced apoptosis in several types of p53+/+ normal human cells, through a caspase-dependent pathway. Together, these results demonstrate that Cox-2 is induced by p53-mediated activation of the Ras/Raf/ERK cascade, counteracting p53-mediated apoptosis. This anti-apoptosis effect may be a mechanism to abate cellular stresses associated with p53 induction

Altered Expression of Fibrosis Genes in Capsules of Failed Ahmed Glaucoma Valve Implants

<div><p>Purpose</p><p>Ahmed glaucoma valve (AGV) implant is an aqueous shunt device used to control intraocular pressure in glaucoma. Implant failure results from impervious encapsulation of the shunt plate causing increased hydraulic resistance and raised intraocular pressure. We hypothesized that deregulation of fibrosis pathway contributes to capsular resistance. We tested this by studying fibrosis related gene expression in failed AGV implants.</p><p>Methods</p><p>Differential gene expression was examined in failed AGV capsules and compared to normal control tenon. Following total RNA extraction, 84 key genes in fibrosis pathway were examined by real-time PCR using RT² Profiler PCR Array. Relative gene expression was calculated using ΔΔC_t method. Gene specific TaqMan assays were used to validate select genes with ≥2 fold differential expression in the array expression profile.</p><p>Results</p><p>We observed differential expression in several genes in the fibrosis pathway. Almost half (39/84) of examined genes showed ≥2 fold differential expression in majority of capsules examined on the array. TaqMan assays for select genes including CCN2 (CTGF), THBS1, SERPINE1, THBS2, COL3A1, MMP3, and IL1A in an increased validation sample set showed significant changes in expression (p value from <0.001 to 0.022) at a high frequency in concurrence with our array results.</p><p>Conclusions</p><p>Pathway-focused analyses identified candidate genes with altered expression providing molecular evidence for deregulation of the fibrosis pathway in AGV failure.</p></div

Validation of differential gene expression by TaqMan Gene expression assays.

<p>The differential gene expression was statistically significant (p value from <0.001 to 0.022) by non-parametric Mann-Whitney U test except for IFNG and IL13RA2.</p

Demographic data and clinical parameters of patients.

<p>Demographic data and clinical parameters of patients.</p

Non-supervised hierarchical clustering of normalized genes examined by PCR analysis.

<p>Non-supervised hierarchical clustering of normalized genes examined by PCR analysis.</p

Differential expression of genes identified by Human Fibrosis PCR-Array analysis in failed AGV capsules.

<p>Capsules 2, 5 and 6 were from patients below 18 years and capsules 1, 3, 4 and 7 were from patients above 18 years of age. Validated gene changes were deregulated in majority of the capsules.</p

Differential gene expression (≥2 fold) in failed AGV capsules determined by fibrosis array and validated by TaqMan gene expression assays.

<p>^aDue to sample limitation and poor amplification analysis was only possible in fewer capsules as indicated.</p><p>Differential gene expression (≥2 fold) in failed AGV capsules determined by fibrosis array and validated by TaqMan gene expression assays.</p

Key processes implicated in AGV failure.

<p>Key processes implicated in AGV failure.</p