τ Regulation of Microtubule-Microtubule Spacing and Bundling

Abstract: τ proteins are microtubule-associated proteins that promote microtubule polymerization in vitro and in vivo. They are a family of neuronal proteins with apparent molecular weights in the range 50,000–68,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recently, a new member of this family has been described and its cDNA has been cloned. It has an apparent molecular weight of 116,000 and has been called high-molecular-weight τ (HMW τ). All the τ proteins are encoded by a single gene, which undergoes complex alternative splicing. In the present study, we have cloned into the baculovirus a cDNA fully encoding HMW τ as well as a truncated cDNA encoding a protein beginning 13 amino acids in front of the τ microtubule-binding domain. HMW τ-recombinant-virus-infected Sf9 cells overexpressed HMW τ, which induced the polymerization of microtubules and the formation of long cellular processes similar to those induced by low-molecular-weight τ (LMW τ) overexpression. Process cross sections revealed a larger spacing (≈35 nm) between microtubules when induced by HMW τ than when induced by LMW τ (≈20 nm). The truncated construct also induces processes, where microtubules were packed far more closely together (≈10 nm). Although branching did not occur in processes induced by intact τs, 10% of the processes induced by the truncated τ protein branched.     

PIG-B: A homemade monophasic cocktail for the extraction of RNA

An inexpensive monophasic reagent has been developed for the extraction of total RNA from cells or tissues. The main ingredients of the reagent arePhenol,Isoamyl alcohol,Guanidinium isothiocyanate, andBeta-mercaptoethanol (PIG-B). The quality and yield of RNA obtained by this reagent is at par with that obtained by TRIzol, an expensive but widely used monophasic reagent available commercially. The complete composition and method of preparation of PIG-B is provided to aid preparation of the reagent in the laboratory.     

Survival of salivary gland cancer stem cells requires mTOR signaling

Advanced salivary gland mucoepidermoid carcinoma (MEC) is a relentless cancer that exhibits resistance to conventional chemotherapy. As such, treatment for patients with advanced MEC is tipically radical surgery and radiotherapy. Facial disfigurement and poor quality of life are frequent treatment challenges, and many patients succumb to loco-regional recurrence and/or metastasis. We know that cancer stem-like cells (CSC) drive MEC tumorigenesis. The current study tests the hypothesis that MEC CSC are sensitive to therapeutic inhibition of mTOR. Here, we report a correlation between the long-term clinical outcomes of 17 MEC patients and the intratumoral expression of p-mTOR (p = 0.00294) and p-S6K1 (p = 0.00357). In vitro, we observed that MEC CSC exhibit constitutive activation of the mTOR signaling pathway (i.e., mTOR, AKT, and S6K1), unveiling a potential strategy for targeted ablation of these cells. Using a panel of inhibitors of the mTOR pathway, i.e., rapamycin and temsirolimus (mTOR inhibitors), buparlisib and LY294002 (AKT inhibitors), and PF4708671 (S6K1 inhibitor), we observed consistently dose-dependent decrease in the fraction of CSC, as well as inhibition of secondary sphere formation and self-renewal in three human MEC cell lines (UM-HMC-1,-3A,-3B). Notably, therapeutic inhibition of mTOR with rapamycin or temsirolimus induced preferential apoptosis of CSC, when compared to bulk tumor cells. In contrast, conventional chemotherapeutic drugs (cisplatin, paclitaxel) induced preferential apoptosis of bulk tumor cells and accumulation of CSC. In vivo, therapeutic inhibition of mTOR with temsirolimus caused ablation of CSC and downregulation of Bmi-1 expression (major inducer of stem cell self-renewal) in MEC xenografts. Transplantation of MEC cells genetically silenced for mTOR into immunodeficient mice corroborated the results obtained with temsirolimus. Collectively, these data demonstrated that mTOR signaling is required for CSC survival, and unveiled the therapeutic potential of targeting the mTOR pathway for elimination of highly tumorigenic cancer stem-like cells in salivary gland mucoepidermoid carcinoma.Subject terms: Cancer stem cells, Cancer stem cells, Head and neck cancer, Oral cancer