Recent studies implicate a role for WD repeat domain, phosphoinositide-interacting 1 (WIPI1) in the biogenesis of melanosomes, cell type-specific lysosome-related organelles. In this study, we determined that WIPI1, an ATG18 homologue that is shown to localize to both autophagosomes and early endosomes, inhibited mammalian target of rapamycin (MTOR) signaling, leading to increased transcription of melanogenic enzymes and the formation of mature melanosomes. WIPI1 suppressed the target of rapamycin complex 1 (TORC1) activity, resulting in glycogen synthase kinase 3␤ inhibition, ␤-Catenin stabilization, and increased transcription of microphthalmia transcription factor and its target genes. WIPI1-depleted cells accumulated stage I melanosomes but lacked stage III-IV melanosomes. Inhibition of TORC1 by rapamycin treatment resulted in the accumulation of stage IV melanosomes but not autophagosomes, whereas starvation resulted in the formation of autophagosomes but not melanin accumulation. Taken together, our studies define a distinct role for WIPI1 and TORC1 signaling in controlling the transcription of melanogenic enzymes and melanosome maturation, a process that is distinct from starvation-induced autophagy.

The dependence of the incidence of radiation-induced cancer on the dose rate of the radiation exposure is a question of considerable importance to the estimation of risk of cancer induction by low-dose-rate radiation. Currently a dose and dose-rate effectiveness factor (DDREF) is used to convert high-dose-rate risk estimates to low dose rates. In this study, the end point of neoplastic transformation in vitro has been used to explore this question. It has been shown previously that for low doses of low-LET radiation delivered at high dose rates, there is a suppression of neoplastic transformation frequency at doses less than around 100 mGy. In the present study, dose-response curves up to a total dose of 1000 mGy have been generated for photons from (125)I decay (approximately 30 keV) delivered at doses rates of 0.19, 0.47, 0.91 and 1.9 mGy/min. The results indicate that at dose rates of 1.9 and 0.91 mGy/min the slope of the induction curve is about 1.5 times less than that measured at high dose rate in previous studies with a similar quality of radiation (28 kVp mammographic energy X rays). In the dose region of 0 to 100 mGy, the data were equally well fitted by a threshold or linear no-threshold model. At dose rates of 0.19 and 0.47 mGy/min there was no induction of transformation even at doses up to 1000 mGy, and there was evidence for a possible suppressive effect. These results show that for this in vitro end point the DDREF is very dependent on dose rate and at very low doses and dose rates approaches infinity. The relative risks for the in vitro data compare well with those from epidemiological studies of breast cancer induction by low- and high-dose-rate radiation.

Melanomas resist conventional chemotherapeutics in part through intrinsic disrespect of apoptotic checkpoint activation. In this study, using an unbiased genome-wide RNAi screen we identified RhoJ and its effector Pak1, as key modulators of melanoma cell sensitivity to DNA damage. We find that RhoJ activates Pak1 in response to drug-induced DNA damage, which then uncouples ATR from its downstream effectors, ultimately resulting in a blunted DNA damage response (DDR). In addition, ATR suppression leads to the decreased phosphorylation of ATF2, and consequent increased expression of the melanocyte survival gene Sox10 resulting in a higher DDR threshold required to engage melanoma cell death. In the setting of normal melanocyte behavior, this regulatory relationship may facilitate appropriate epidermal melanization in response to UV-induced DNA damage. However, pathological pathway activation during oncogenic transformation produces a tumor that is intrinsically resistant to chemotherapy and has the propensity to accumulate additional mutations. These findings identify DNA damage agents and pharmacological inhibitors of RhoJ/PAK1 as novel synergistic agents that can be used to treat melanomas that are resistant to conventional chemotherapies.

gene expresses a plasma membrane protein involved in the transport of the watersoluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.