Biomedical subjects

Yuval Shaked

Publications and source records attributed to Yuval Shaked.

23 records · Page 2Linked to original sources

PubMed2004

Radiation resistance of human melanoma analysed by retroviral insertional mutagenesis reveals a possible role for dopachrome tautomerase.

While melanomas are resistant to the cytotoxic effects of radiotherapy, little is known about the molecular mechanisms underlying this intrinsic resistance. Here, we describe the utilization of retroviral insertional mutagenesis to facilitate the analysis of genetic changes that are associated with radioresistance in human melanoma. A radial growth phase human melanoma cell line, WM35, was infected with a replication-defective amphotropic murine retrovirus and subsequently selected for X-ray radiation-resistant variants. Several radiation-resistant clones were independently isolated and characterized. Interestingly, these clones also displayed resistance to ultraviolet radiation and to the chemotherapeutic drug cis-diamminedichloroplatinum(II) (CDDP). By Northern and Western analyses, we showed that the expression of DOPAchrome tautomerase (DCT), also known as tyrosinase-related protein 2 (TYRP2), an enzyme that functions in eumelanin synthesis, was significantly elevated in the radiation-resistant clones relative to the parental WM35 cells. Moreover, the levels of DCT in a variety of human melanoma cell lines correlated with their relative levels of radioresistance and the enforced expression of DCT conferred increased resistance to UV(B) treatment. An analysis of stress signaling induced by radiation as well other cytotoxic stressors showed that resistance associated with DCT overexpression applied specifically to treatments that activate the ERK/MAPK pathway. Indeed, DCT overexpression in a melanoma cell line resulted in increased ERK activity. Moreover, ectopic expression of dominant-active MEK in this melanoma cell line conferred UV(B) resistance suggesting that the ERK/MAPK pathway downstream of DCT may play a critical role in radiation and drug resistance. Overall, given that each gamma- and UV(B)-resistant cell line also exhibited resistance to CDDP and that CDDP-resistant clones showed increased resistance to UV(B) irradiation, these results suggest a common mechanism underlying radio- and chemoresistance, which is mediated by DCT expression.

Cell Line, Tumor↗

PubMed2003

Copper binding to PrPC may inhibit prion disease propagation.

Although it has been well established that PrP(C), the normal isoform of PrP(Sc), is a copper-binding protein, the role of this metal in the function of PrP(C) as well as in prion disease pathology remains unclear. Here, we show that when scrapie-infected neuroblastoma cells were cultured in the presence of copper, the accumulation of PrP(Sc) in these cells was markedly reduced. In addition, our results indicate that when normal neuroblastoma cells were cultured in the presence of copper ions, they could no longer bind and internalize PrP(Sc). In another set of experiments, copper was added to the drinking water of normal and scrapie-infected hamsters. Our results show that administration of copper to normal hamsters induced cerebellar PrP(C) accumulation. Most important, a significant delay in prion disease onset was observed when scrapie-infected hamsters were treated with copper. As shown before for neuroblastoma cells, also in vivo most of the copper-induced accumulation of PrP(C) was intracellular. We hypothesized that PrP(C) internalization by copper may hinder PrP(Sc) interaction with this molecule, and thereby affect prion disease propagation.

Animals↗

PubMed2003

Maximum tolerable dose and low-dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells.

There is growing evidence that vasculogenesis (progenitor cell-derived generation of new blood vessels) is required for the growth of some neoplastic diseases. Here we show that the administration of cyclophosphamide (CTX) at the maximum tolerable dose with 21-day breaks or at more frequent low-dose (metronomic) schedules have opposite effects on the mobilization and viability of circulating endothelial progenitors (CEPs) in immunodeficient mice bearing human lymphoma cells. Animals treated with the maximum tolerable dose CTX experienced a robust CEP mobilization a few days after the end of a cycle of drug administration, and tumors rapidly became drug resistant. Conversely, the administration of metronomic CTX was associated with a consistent decrease in CEP numbers and viability and with more durable inhibition of tumor growth. Our findings suggest that metronomic low-dose chemotherapy regimens are particularly promising for avoiding CEP mobilization and, hence, to potentially reduce vasculogenesis-dependent mechanisms of tumor growth.

Animals↗

PubMed2002

Doppel and PrP(C) do not share the same membrane microenvironment.

Doppel is a paralog of the normal prion protein, PrP(C). It has been suggested that Doppel can compensate for the absence of PrP(C) in PrP(0/0) mice. In this work, we tested whether Doppel and PrP(C) share the same cell location, thereby sharing the same neighboring cell components, probably required to share the same cell function. Our results show that, at detergent conditions in which membrane rafts were intact, neither PrP(C) and Doppel co-immunoprecipitate with the appropriate antibodies, nor was Doppel retained by a Cu(2+)IMAC resin, as PrP(C) does. This indicates that, although Doppel is a raft-associated protein as is PrP(C), both proteins are not present in the same membrane microenvironment, and they probably do not perform the same function.

Animals↗

PubMed2002

The binding of prion proteins to serum components is affected by detergent extraction conditions.

As many GPI anchored proteins, PrP(C) and its abnormal conformer PrP(Sc), are inserted into membrane microdomains known as rafts. Upon raft disruption, PrP(C) becomes soluble, while PrP(Sc) aggregates into insoluble structures. It was recently published that, as opposed to PrP(C), PrP(Sc), as well as its protease resistant core PrP27-30, can bind specifically to plasminogen and other serum components. These findings were suggested to have important physiological implications in transmissible spongiform encephalopathies (TSE) diagnosis and pathogenesis. In this work, we show that the binding of PrP(Sc) or PrP 27-30 to serum proteins occurs only at specific detergent combinations, in which disease associated PrPs are present in aggregated structures. At detergent conditions in which rafts are intact, it is actually PrP(C.) that binds to blood proteins, albeit not directly, but through neighboring rafts components. Our results therefore indicate that the binding of PrP(Sc) to blood components has no physiological relevance.

Animals↗