Myoepithelial molecular markers in human breast carcinoma PMC42-LA cells are induced by extracellular matrix and stromal cells

The microenvironment plays a key role in the cellular differentiation of the two main cell lineages of the human breast, luminal epithelial, and myoepithelial. It is not clear, however, how the components of the microenvironment control the development of these cell lineages. To investigate how lineage development is regulated by 3-D culture and microenvironment components, we used the PMC42-LA human breast carcinoma cell line, which possesses stem cell characteristics. When cultured on a two-dimensional glass substrate, PMC42-LA cells formed a monolayer and expressed predominantly luminal epithelial markers, including cytokeratins 8, 18, and 19; E-cadherin; and sialomucin. The key myoepithelial-specific proteins alpha-smooth muscle actin and cytokeratin 14 were not expressed. When cultured within Engelbreth-Holm- Swarm sarcoma-derived basement membrane matrix (EHS matrix), PMC42-LA cells formed organoids in which the expression of luminal markers was reduced and the expression of other myoepithelial-specific markers (cytokeratin 17 and P-cadherin) was promoted. The presence of primary human mammary gland fibroblasts within the EHS matrix induced expression of the key myoepithelial-specific markers, alpha-smooth muscle actin and cytokeratin 14. Immortalized human skin fibroblasts were less effective in inducing expression of these key myoepithelial-specific markers. Confocal dual-labeling showed that individual cells expressed luminal or myoepithelial proteins, but not both. Conditioned medium from the mammary fibroblasts was equally effective in inducing myoepithelial marker expression. The results indicate that the myoepithelial lineage is promoted by the extracellular matrix, in conjunction with products secreted by breast-specific fibroblasts. Our results demonstrate a key role for the breast microenvironment in the regulation of breast lineage development.

Antitumor activity and underlying mechamisms of ganopoly, the refined polysaccharides extracted from ganodrema lucidum, in mice

Ganopoly is an aqueous polysaccharide fraction extracted from G. lucidum by patented biochemical technique and has been marketed as an over-the-counter product for chronic diseases including cancer and hepatopathy in many Asian countries. This study was undertaken to explore the anti-tumour effect and the underlying mechanisms of Ganopoly in mice and human tumor cell lines. The maximum tolerateddose (MTD) of Ganopoly in mice was estimated to be 100 mg/kg from a pilot study. Treatment of mice with oral Ganopoly for 10 days significantly reduced the tumour weight of sarcoma-180 in a dose-dependent manner, with inhibition rates of 32.3, 48.2 and 84.9% and growth delays of 1.5, 3.5, and 13.1 days at 20, 50, and 100 mg/kg, respectively. Incubation of Ganopoly at 0.05-1.0 mg/ml for 48 hours showed little or negligible cytotoxicity against human tumor CaSki, SiHa, Hep3B, HepG2, HCT116, HT29, and MCF7 cells in vitro. In contrast, 10 mg/ml of Ganopoly caused significant cytotoxicity in all tumour cells tested except MCF7, with marked apoptotic effects observed in CaSki, HepG2, and HCT116 cells, as indicated by nuclear staining and DNA fragmentation. In addition, Ganopoly enhanced concanavalin A-stimulated proliferation of murine splenocytes by 35.3% at 10 mg/ml, and stimulated the production of nitric oxide in thioglycollate-primed murine peritoneal macrophages in a concentration-dependent manner over 0.05-10 mg/ml. Addition of Ganopoly at 1 mg/ml to murine peritoneal macrophages also potentiated lipopolysaccharide-induced nitric oxide production by 64.2%. Treatment of healthy mice or mice bearing sarsoma-180 with oral Ganopoly over 20-100 mg/kg for 7 day significantly increased the expression of both TNF-α and IFN-γ (at both mRNA and protein levels) in splenocytes in a dose-dependent manner. Moreover, treatment of Ganopoly over 20-100 mg/kg significantly increased cytotoxic T lymphocyte cytotoxicity and NK activity in mice. The overall findings indicated that Ganopoly had antitumor activity with a broad spectrum of immuno-modulating activities and may represent a novel promising immunotherapeutic agent in cancer treatment.

Rapid detection of SMARCB1 sequence variation using high resolution melting

Background : Rhabdoid tumors are rare cancers of early childhood arising in the kidney, central nervous system and other organs. The majority are caused by somatic inactivating mutations or deletions affecting the tumor suppressor locus SMARCB1 [OMIM 601607]. Germ-line SMARCB1 inactivation has been reported in association with rhabdoid tumor, epitheloid sarcoma and familial schwannomatosis, underscoring the importance of accurate mutation screening to ascertain recurrence and transmission risks. We describe a rapid and sensitive diagnostic screening method, using high resolution melting (HRM), for detecting sequence variations in SMARCB1. Methods : Amplicons, encompassing the nine coding exons of SMARCB1, flanking splice site sequences and the 5' and 3' UTR, were screened by both HRM and direct DNA sequencing to establish the reliability of HRM as a primary mutation screening tool. Reaction conditions were optimized with commercially available HRM mixes. Results : The false negative rate for detecting sequence variants by HRM in our sample series was zero. Nine amplicons out of a total of 140 (6.4%) showed variant melt profiles that were subsequently shown to be false positive. Overall nine distinct pathogenic SMARCB1 mutations were identified in a total of 19 possible rhabdoid tumors. Two tumors had two distinct mutations and two harbored SMARCB1 deletion. Other mutations were nonsense or frame-shifts. The detection sensitivity of the HRM screening method was influenced by both sequence context and specific nucleotide change and varied from 1: 4 to 1:1000 (variant to wild-type DNA). A novel method involving digital HRM, followed by re-sequencing, was used to confirm mutations in tumor specimens containing associated normal tissue. Conclusions : This is the first report describing SMARCB1 mutation screening using HRM. HRM is a rapid, sensitive and inexpensive screening technology that is likely to be widely adopted in diagnostic laboratories to facilitate whole gene mutation screening.

Impact of HIV-associated conditions on mortality in people commencing anti-retroviral therapy in resource limited settings

Objectives: To identify associations between specific WHO stage 3 and 4 conditions diagnosed after ART initiation and all cause mortality for patients in resource-limited settings (RLS).

Design, Setting: Analysis of routine program data collected prospectively from 25 programs in eight countries between 2002 and 2010.

Subjects, Participants: 36,664 study participants with median ART follow-up of 1.26 years (IQR 0.55–2.27).

Outcome Measures: Using a proportional hazards model we identified factors associated with mortality, including the occurrence of specific WHO clinical stage 3 and 4 conditions during the 6-months following ART initiation.

Results: There were 2922 deaths during follow-up (8.0%). The crude mortality rate was 5.41 deaths per 100 person-years (95% CI: 5.21–5.61). The diagnosis of any WHO stage 3 or 4 condition during the first 6 months of ART was associated with
increased mortality (HR: 2.21; 95% CI: 1.97–2.47). After adjustment for age, sex, region and pre-ART CD4 count, a diagnosis of extrapulmonary cryptococcosis (aHR: 3.54; 95% CI: 2.74–4.56), HIV wasting syndrome (aHR: 2.92; 95%CI: 2.21 -3.85), nontuberculous mycobacterial infection (aHR: 2.43; 95% CI: 1.80–3.28) and Pneumocystis pneumonia (aHR: 2.17; 95% CI 1.80–3.28) were associated with the greatest increased mortality. Cerebral toxoplasmosis, pulmonary and extra-pulmonary
tuberculosis, Kaposi’s sarcoma and oral and oesophageal candidiasis were associated with increased mortality, though at lower rates.

Conclusions: A diagnosis of certain WHO stage 3 and 4 conditions is associated with an increased risk of mortality in those initiating ART in RLS. This information will assist initiatives to reduce excess mortality, including prioritization of resources for
diagnostics, therapeutic interventions and research.