Background: Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.
Squamous cell carcinomas (SCCs) originate in stratified epithelia, with a small subset becoming metastatic. Epithelial stem cells are targets for driver mutations that give rise to SCCs, but it is unknown whether they contribute to oncogenic multipotency and metastasis. We developed a mouse model of SCC by targeting two frequent genetic mutations in human SCCs, oncogene Kras G12D activation and Smad4 deletion, to mouse keratin 15-expressing (K15 + ) stem cells. We show that transgenic mice developed multilineage tumors, including metastatic SCCs. Among cancer stem cell-enriched (CSC-enriched) populations, those with increased side population (SP) cells correlated with epithelial-mesenchymal transition (EMT) and lung metastasis. We show that microRNA-9 (miR-9) contributed to SP expansion and metastasis, and miR-9 inhibition reduced the number of SP cells and metastasis. Increased miR-9 was detected in metastatic human primary SCCs and SCC metastases, and miR-9-transduced human SCC cells exhibited increased invasion. We identified α-catenin as a predominant miR-9 target. Increased miR-9 in human SCC metastases correlated with α-catenin loss but not E-cadherin loss. Our results demonstrate that stem cells with Kras G12D activation and Smad4 depletion can produce tumors that are multipotent and susceptible to EMT and metastasis. Additionally, tumor initiation and metastatic properties of CSCs can be uncoupled, with miR-9 regulating the expansion of metastatic CSCs. IntroductionSquamous cell carcinomas (SCCs) are derived from stratified epithelia present within the skin and oral cavity. A subset of aggressive SCCs become metastatic and lead to metastasis-associated death. The rate of metastasis in skin SCCs ranges from 0.1% to 10% (1), with poorly differentiated tumors and those with greater vertical tumor thickness having an increased risk of metastasis (2). Genetic alterations and intrinsic tumor cell properties controlling SCC metastasis are largely unknown. Genetically engineered mice provide a powerful tool for dissecting driver mutations that contribute to SCC initiation and metastasis. To date, very few genetic mutations causing spontaneous SCC formation and metastasis have been found, particularly metastasis to the lung, which is the leading cause of SCC-associated death (3). Mice with a Smad4 deletion in stratified epithelia develop spontaneous SCCs in the skin, oral cavity, and forestomach (4-6). Among these models, oral SCCs metastasize to lymph nodes (4), whereas skin and forestomach SCCs do not metastasize (5, 6).Because stratified epithelia undergo constant self-renewal and rapid turnover, it is believed that driver mutations for SCCs must initially occur in resident stem cells that renew these epithelia throughout life. In mouse skin, the hair follicle bulge harbors
In vitiligo, the autoimmune destruction of epidermal melanocytes produces white spots that can be repigmented by melanocyte precursors from the hair follicles, following stimulation with UV light. We examined by immunofluorescence the distribution of melanocyte markers (C-KIT, DCT, PAX3, and TYR) coupled with markers of proliferation (KI-67) and migration (MCAM) in precursors and mature melanocytes from the hair follicle and the epidermis of untreated and narrow band UVB (NBUVB)-treated human vitiligo skin. NBUVB was associated with a significant increase in the number of melanocytes in the infundibulum and with restoration of the normal melanocyte population in the epidermis, which was lacking in the untreated vitiligo. We identified several precursor populations (melanocyte stem cells, melanoblasts, and other immature phenotypes), and progressively differentiating melanocytes, some with putative migratory and/or proliferative abilities. The primary melanocyte germ was present in the untreated and treated hair follicle bulge, whereas a possible secondary melanocyte germ composed of C-KIT+ melanocytes was found in the infundibulum and interfollicular epidermis of UV-treated vitiligo. This is an exceptional model for studying the mobilization of melanocyte stem cells in human skin. Improved understanding of this process is essential for designing better treatments for vitiligo, ultimately based on melanocyte stem cell activation and mobilization.
The Bcl-2 family is important in modulating sensitivity to anticancer drugs in many cancers, including melanomas. The BH3 mimetic ABT-737 is a potent small molecule inhibitor of the anti-apoptotic proteins Bcl-2/Bcl-X(L)/Bcl-w. In this report, we examined whether ABT-737 is effective in killing melanoma cells in combination with the proteasome inhibitor MG-132, and further evaluated the mechanisms of action. Viability, morphological, and Annexin V apoptosis assays showed that ABT-737 alone exhibited little cytotoxicity, yet it displayed strong synergistic lethality when combined with MG-132. In addition, the detection of Bax/Bak activation indicated that the combination treatment synergistically induced mitochondria-mediated apoptosis. Furthermore, mechanistic analysis revealed that this combination treatment induced expression of the pro-apoptotic protein Noxa- and caspase-dependent degradation of the anti-apoptotic protein, Mcl-1. Finally, siRNA-mediated inhibition of Mcl-1 expression significantly increased sensitivity to ABT-737 in these cells, and knocking down Noxa expression protected the cells from cytotoxicity induced by the combination treatment. These findings demonstrate that ABT-737 combined with MG-132 synergistically induced Noxa-dependent mitochondrial-mediated apoptosis. In summary, this study indicates promising therapeutic potential of targeting anti-apoptotic Bcl-2 family members in treating melanoma, and it validates rational molecular approaches that target anti-apoptotic defenses when developing cancer treatments.
Hyperthermia has been revived as a promising approach for cancer treatment. To understand the underlying mechanisms of hyperthermic killing of cancer cells, we examined the cytotoxic effects of hyperthermia on various skin cancer cell lines using cell viability, morphological analyses, and caspase activation assays. Hyperthermia induced cytotoxicity in a time- and temperature-dependent manner. At middle dose/time combinations, heat-induced apoptosis, whereas at higher doses, necrosis was the mechanism of cell death. To investigate the mechanisms of hyperthermia-induced apoptosis, we examined the activation of extrinsic (Caspase 8) and intrinsic (Caspase 9) apoptotic pathways. Hyperthermia did not activate Caspases 8 or 9, but did activate Caspase 3/7, suggesting a non-conventional apoptotic pathway. Last, analysis of Grp78 expression and Caspase 12 or 4 activation indicated that hyperthermia induced endoplasmic reticulum-mediated apoptosis. Thus, hyperthermia induced apoptosis in two types of skin cancer cells through endoplasmic reticulum-mediated apoptosis and not through the classical intrinsic or extrinsic apoptosis pathways. Hyperthermia may be a promising treatment for basal cell carcinoma and melanoma, bypassing the antiapoptotic defenses concentrated in the intrinsic and extrinsic apoptosis pathways. These results also raise the possibility that heat may be combined with other approaches for induction of apoptosis to achieve synergistic killing of skin cancers.
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