Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by onedimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.For several years, work in our laboratory has been concerned with the characterization of an abundant cytoplasmic protein migrating with an apparent Mr of 85,000 (85K protein) in sodium dodecyl sulfate (SDS)-polyacrylamide gels. We described the purification and preliminary characterization of the denatured protein (22) and established that its synthesis and turnover are regulated when L929 cells are maintained in the absence of glucose (14,21). Based on mobility in SDS-polyacrylamide gels and one-dimensional partial proteolytic peptide maps, we concluded that this protein is identical with one of the pp60src-associated proteins (23). Subsequently, we showed that synthesis of this protein is induced, along with several others, by exposure to elevated temperatures (15,21,24), i.e., that it is a heat shock protein (hsp85).The heat shock proteins are synthesized by a wide variety of cells after exposure to elevated temperatures. Although it has been suggested that these highly conserved proteins may participate in opposing the lethal (12,22,26,35) or metabolic (10, 21) effects of hyperthermia, their function is still essentially unknown. Since certain functions might be excluded or suggested by a unique intracellular location, e...

Two monoclonal antibodies have been produced against the human 85,000-molecular-weight heat shock protein (hsp85). One of these, 16F1, cross-reacts with the murine homolog and is shown by peptide map immunoblots to be directed against an epitope different from that recognized by the other monoclonal antibody, 9D2. Both monoclonal antibodies recognize only a single Mr-85,000 species in two-dimensional immunoblots. Immunoprecipitation did not reveal an association of this heat shock protein with any other protein in HeLa cells. Immunoperoxidase staining showed a purely cytosolic distribution at both light and electron microscopic levels and no association with membranes, mitochondria, or other organelles. The 9D2 monoclonal and a polyclonal antimurine hsp85 antibody were used to identify the antigens and to quantitate their levels in a variety of normal tissues by immunoautoradiography. Relative abundance in the various tissues as determined by Coomassie blue staining correlates reasonably well with the immunoreactivity. Testis and brain, for example, have high hsp85 levels, whereas heart and skeletal muscle have little or none. The Mr-85,000 sodium dodecyl sulfate-polyacrylamide gel band in testis and brain lysates was further confirmed to be hsp85 by one-dimensional partial proteolytic peptide mapping. Based on these data and our previous observations showing that synthesis and levels of the protein are altered by depriving cultured cells of glucose, we speculate that intracellular hsp85 levels depend on differences in the intermediary metabolism of glucose in the various tissues. Furthermore, it appears that high basal levels of this heat shock protein may not necessarily protect cells against heat shock, since testis is one of the most heat-sensitive tissues and has the highest hsp85 level.

A transferrin-doxorubicin conjugate exhibited greatly increased cytotoxicity relative to unconjugated doxorubicin toward a variety of cultured tumor cell lines. An L929 cell line selected for doxorubicin resistance was as sensitive to the transferrin-doxorubicin conjugate as was the parental unselected line. Quantitative measurements of doxorubicin fluorescence in single L929 cells showed that uptake was similar in amount when cells were exposed to equivalent concentrations of doxorubicin presented either free or as the transferrin-doxorubicin conjugate. However, unconjugated drug fluorescence was distributed in membranes, cytoplasm and nucleus, whereas conjugate fluorescence was confined mainly to the cytoplasmic compartment. In as much as NADPH-dependent free radical formation is a known mechanism of doxorubicin cytotoxicity, localization in the vicinity of NADPH production might facilitate this cytotoxic pathway. Neither cytotoxicity nor uptake of the conjugate quantified by doxorubicin fluorescence was significantly blocked by excess free transferrin, and the conjugate was not concentrated in the plasma membrane at 4 degrees C. These findings suggest that conjugate internalization is not entirely dependent on transferrin receptor binding.

We have shown that, whereas argon ion laser irradiation alone is not cytocidal for L929 cells, it greatly increases the cytotoxicity of intracellular doxorubicin. The present study showed that light enhancement of doxorubicin cytotoxicity was not restricted to stock L929 cells, but could also be demonstrated using L929 cells selected for doxorubicin resistance and several standard cell lines that are relatively resistant to doxorubicin prior to selection. Light-enhanced cytotoxicity resulted in extensive nuclear DNA loss and was strongly inhibited by anoxia. These findings suggest that the mechanism by which light exposure enhances doxorubicin cytotoxicity involves DNA damage by intranuclear generation of reactive oxygen species.

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