Human melanoma cells under endoplasmic reticulum stress acquire resistance to microtubule-targeting drugs through XBP-1-mediated activation of Akt.

Past studies have shown that melanoma cells have largely adapted to endoplasmic reticulum (ER) stress. In this study, we report that melanoma cells under ER stress are more resistant to apoptosis induced by the microtubule-targeting chemotherapeutic drugs, docetaxel and vincristine, and this is, at least in part, due to activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway mediated by the X-box-binding protein 1 (XBP-1) axis of the unfolded protein response. Treatment with the ER stress-inducer tunicamycin (TM) or thapsigargin before the addition of docetaxel or vincristine reduced the levels of apoptosis induced by the drugs. This was associated with inhibition of mitochondrial release of apoptogenic proteins and activation of Bax and Bak. Induction of ER stress resulted in the rapid activation of the PI3K/Akt pathway that seemed to be important in antagonizing docetaxel and vincristine, in that inhibition of Akt blocked the effect of pretreatment with TM on apoptosis induced by the drugs. Neither docetaxel nor vincristine triggered ER stress in melanoma cells, but the basal activity of XBP-1 signaling seemed to play a role in the protection against the drugs because small interfering RNA knockdown of XBP-1 enhanced docetaxel- and vincristine-induced apoptosis. In addition, inhibition of XBP-1 decreased the constitutive levels of activation of Akt and blocked the activation of Akt induced by TM. Taken together, these results identify activation of the PI3K/Akt pathway by XBP-1-mediated signaling of the unfolded protein response as a resistance mechanism against docetaxel and vincristine in melanoma cells under ER stress.

Activation of Jun N-terminal kinase is a mediator of vincristine-induced apoptosis of melanoma cells.

The molecular changes involved in the induction of apoptosis by vincristine in melanoma have not yet been well defined. Two human melanoma cell lines showing moderate (Mel-RM) and high (IgR3) sensitivity to vincristine were selected from a panel of eight melanoma lines for analysis. Induction of apoptosis was caspase dependent, and was associated with increases in mitochondrial membrane permeability. Vincristine upregulated the expression of Bax, Bak, PUMA, Noxa, p53 and p21 proteins, and downregulated and/or phosphorylated the Bcl-2 protein. Inhibitors of the Jun N-terminal kinase (JNK), but not p38 mitogen-activated protein kinase, significantly inhibited vincristine-induced apoptosis in both IgR3 and Mel-RM cells. In addition, vincristine induced phosphorylation and reduction in Bcl-2 was prevented by an inhibitor of JNK. Downregulation of mRNA for p53, PUMA or Bim by RNA interference had little or no influence on vincristine-induced apoptosis in IgR3 cells. In addition, silencing Bim mRNA did not affect vincristine-induced apoptosis in Mel-RM cells. These results suggest that vincristine-induced apoptosis of at least some melanoma cell lines is dependent on the activation of JNK. The results are consistent with the phosphorylation of Bcl-2 protein, resulting in the activation of Bax/Bak, release of cytochrome c from the mitochondria and the resulting activation of caspases.

Determination of transcription factors and their possible roles in the regulation of Pax3 gene expression in the mouse B16 F1 melanoma cell line.

The objective of this study was to determine which transcription factors regulate the expression of the Pax3 gene in the mouse B16 F1 melanoma cell line. The results showed that the -14 kilobase pair (kbp) Pax3 promoter, but not the -1.6 kbp Pax3 promoter, promoted Pax3 gene expression in B16 cells. Comparison of the sequence of the -14 kbp human Pax3 promoter with mouse Pax3 promoters indicated that homology sequences were located between -6.9 and -5.8 kbp, and also that the 1.1 kbp fragment (between -6.9 and -5.8 kbp), linked -1.6 kbp proximal to the Pax3 promoter [plasmid PGPax3PIV (N6.9/5.8) delta SST Lacz], could mimic the functions of plasmid PGPax3 -14(N-1.6) Lacz. Mutations of the core binding elements of either Pax3 site I or II or both sites I and II reduced significantly the beta-galactosidase (beta-gal) activity in the cells. However, mutations of the core binding sequences of site A or B increased significantly the beta-gal activity in the cells. Biochemistry analysis demonstrated that POU transcription factors (Oct-1 and Brn-2) bind to the specific binding elements of both sites I and II to stimulate Pax3 gene expression, whereas the TALE homeodomain-containing proteins (Pbx and Prep1) bind with the core binding sequences of sites A and B to repress the expression of the Pax3 gene in B16 cells.

Optimisation of in vitro culture conditions in B6CBF1 mouse embryos.

This study was undertaken to investigate the effects of three media, volume and type of oil and frequency of observation on the in vitro development of mouse zygotes. B6CBF1 female mice (4 to 6 wk old) were superovulated using PMSG/hCG and mated with a proven fertile male of the same strain. Putative zygotes with polar bodies were collected from the oviducts of mated mice, 25-28 h after hCG injection, and were cultured in vitro. Embryo development was evaluated at either 96 h and 120 h or every 24 h for 120 h. The results obtained showed that the CZB medium was better than the KSOM and HCO3HTF media, and the use of 1 mL of paraffin oil was better than the use of 0.5 mL of paraffin oil. The effect of paraffin oil and mineral oil on embryo development was examined and the results indicated that the use of paraffin oil was better than the use of mineral oil. Repeated observations did not influence the proportion of embryos developing to blastocysts.

Effects of paternal heat stress on the in vivo development of preimplantation embryos in the mouse.

The objective of this study was to examine the effect of paternal heat stress on the in vivo development of preimplantation embryos in the mouse. Synchronised B6CBF1 female mice were mated either to a control male mouse or to one that had been exposed at 7, 21 or 35 days previously, for 24 h to an ambient temperature of 36+/-0.3 degrees C and 66+/-5.6% relative humidity. Embryos were collected from the oviducts of mice at 14-16 h, 34-39 h or 61-65 h after mating or from the uterus at 85-90 h after mating and their developmental status was evaluated morphologically. The number of cells within blastocysts was also determined using bisbenzimide-propidium iodide staining. Paternal heat stress 7 days before mating reduced the proportion of embryos developing from 4-cell (4-C) to morulae (M), hatched blastocysts, total blastocysts and the number of inner cell mass (ICM) and trophectoderm (TE) cells in the blastocyst. Paternal heat stress 21 days prior to mating reduced the proportion of 2-C and 4-C to M embryos with no embryos developing to blastocysts. There were also increases in the number of 1-C and abnormal embryos recorded at this time. Paternal heat stress 35 days before mating decreased the proportion of 2-C embryos, expanded blastocysts and ICM and TE cells in the blastocyst. These results support previous work demonstrating that both the sperm in the epididymis and germ cells in the testis are susceptible to damage by environmental heat stress, with spermatocytes being the most vulnerable. This study also demonstrates that subtle effects on the male such as a short exposure to elevated environmental temperatures can translate to quite profound paternal impacts on early embryo development.