Evidence for segmental gene conversion between a cognate hsp 70 gene and the temperature-sensitively transcribed hsp70 genes of Trypanosoma brucei

The protozoan parasite Trypanosoma brucei expresses several heat shock proteins of 70 kDa (hsp70). We show that, from 5' to 3', a diverged cognate hsp70 gene (gene 1) is separated by about 6 kb of DNA from a cluster of five identical hsp70 genes (genes 2-6). The hsp70 cognate gene has a predicted open reading frame of 676 amino-acids. The steady-state mRNA levels of gene 1 are unaffected by temperature shifts up to 42 degrees C. Hsps of diverse organisms share several fully conserved amino acid domains in the N-terminal region of the hsp70 proteins. These conserved amino acid domains are also observed in the T. brucei hsp70 genes 1-6. However they are, in contrast to the heat shock genes of other eukaryotes, encoded by nucleotide sequence blocks that are identical in all six hsp70 genes. These conserved domains, located in the 5' coding region, range in size from several to hundreds of nucleotides and are separated by highly diverged nucleotide sequences. The nucleotide sequence conservation between hsp70 gene 1 and hsp70 genes 2-6 indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino acid sequence conservation.     

Reciprocal modification of Fas activation and stress protein response decides apoptosis or resistance development of cells

Activation of heat shock factor (HSF)-1 DNA binding and heat shock protein (hsp)-70 expression enable resistance of cells to various forms of stress and maintain cell survival. Fas, a membrane-bound protein, is a central pro-apoptotic factor. Its activation leads to a cascade of events resulting in programmed cell death. Herein, these two mechanisms with contrary functions, promoting either cell survival or death, were addressed for their potential to inhibit each other's activation. Induction of Fas-mediated signalling was followed by a rapid decrease of HSF1 DNA binding and inducible hsp70 expression. Inhibition of HSF1 DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS-signalling. These effects of Fas-activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1)-inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase of apoptosis rates from 20% to 50% in response to heat stress. When analyzing Fas-mediated apoptosis in the presence of HSF1/hsp70 activation, decreased apoptosis rates were detected with induced expression of hsp70 but not with activation of HSF1-DNA binding alone. Thus, we conclude that inhibition of the HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis.     

Bleomycin induces the hsp 70 heat shock promoter in cultured cells

Bleomycin-induced lung disease is characterized by cell injury followed by fibroblast proliferation. Cells respond to injury by synthesizing a family of heat shock proteins. These proteins are critical to cell survival, and those of the 70,000 MW group (hsp 70) are essential for cell division and proliferation. To evaluate the effect of bleomycin on heat shock gene expression, we transfected a gene construct containing the hsp 70 heat shock gene promoter into fibroblasts. Doses of bleomycin, which have previously been shown to augment lung fibroblast proliferation, induce the hsp 70 heat shock promoter in the transfected cells. Bleomycin did not induce the expression of a non-hsp promoter placed in cells as a control of nonspecific gene activation. These observations suggest that bleomycin exposure may cause significant alterations in important DNA promoter regions such as the hsp 70 promoter and point to new ways to assess bleomycin-induced changes in cells.     

Heat shock protein derived from a non-autologous tumour can be used as an anti-tumour vaccine

Antigenic cross-reactivity between certain tumours has allowed the development of more widely applicable, major histocompatibility complex-disparate (allogeneic) whole-cell vaccines. This principle should also allow heat shock proteins (hsp) derived from certain tumours (and carrying cross-reactive antigens) to be used as vaccines to generate anti-tumour immunity in a range of cancer patients. Here, hsp70 derived from gp70-antigen+ B16 melanoma generated cytotoxic-T-lymphocyte-mediated immune protection in BALB/c mice against challenge with gp70-antigen+ CT26 colorectal tumour cells. Using ovalbumin as a model tumour antigen, it is shown that hsp70 enhances peptide re-presentation by dendritic cells via class I over equimolar whole ovalbumin antigen. However, while transfection of tumour cells with inducible hsp70 increases hsp yield from tumours, it does not enhance antigen recognition via purified hsp70 nor via whole cells or their lysate.     

Heat‐shock–mediated conditional regulation of hedgehog/gli signaling in zebrafish

Background: Hedgehog (Hh) signaling is required for embryogenesis and continues to play key roles postembryonically in many tissues, influencing growth, stem cell proliferation, and tumorigenesis. Systems for conditional regulation of Hh signaling facilitate the study of these postembryonic Hh functions. Results: We used the hsp70l promoter to generated three heat‐shock–inducible transgenic lines that activate Hh signaling and one line that represses Hh signaling. Heat‐shock activation of these transgenes appropriately recapitulates early embryonic loss or gain of Hh function phenotypes. Hh signaling remains activated 24 hr after heat shock in the Tg(hsp70l:shha‐EGFP) and Tg(hsp70l:dnPKA‐BGFP) lines, while a single heat shock of the Tg(hsp70l:gli1‐EGFP) or Tg(hsp70l:gli2aDR‐EGFP) lines results in a 6‐ to 12‐hr pulse of Hh signal activation or inactivation, respectively. Using both in situ hybridization and quantitative polymerase chain reaction, we show that these lines can be used to manipulate Hh signaling through larval and juvenile stages. A ptch2 promoter element was used to generate new reporter lines that allow clear visualization of Hh responding cells throughout the life cycle, including graded Hh responses in the embryonic central nervous system. Conclusions: These zebrafish transgenic lines provide important new experimental tools to study the embryonic and postembryonic roles of Hh signaling. Developmental Dynamics 242:529–539, 2013. © 2013 Wiley Periodicals, Inc.