Overexpression of P-glycoprotein in heat-and/or drug-resistant hepatoma variants

We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68.Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine. The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 g/ml colchicine (termed ashighly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance. No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA. Stableheat-resistant variants of clone 2 were also isolated, which showed increaseddrug resistance to several drugs, i.e. they becamemoderately multidrug-resistant. This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants). The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heatresistant, colchicine selected, highly drug-resistant variants. Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional. Verapamil increased doxorubicin retention and cytotoxicity significantly. Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against enviromental stress such as heat.Abbreviations hsp heat-shock protein - MDR multidrug resistance - P-gp P-glycoprotein     

The effect of extracellular Ca2+ and temperature on the induction of the heat-shock and glucose-regulated proteins in hamster fibroblasts

The expression of two stress-inducible protein families was examined in hamster fibroblast cells. These are the heat-shock and glucose-regulated proteins which have been shown to be highly inducible by heat and glucose-starvation, respectively. Our studies here demonstrate that the two sets of proteins can be induced simultaneously or separately. The enhanced synthesis of one set of proteins apparently does not affect the level of expression of the other set. We further show that pre-incubation of these fibroblast cells in calcium-free medium does not inhibit the synthesis of the 70 and 72-kilodalton heat-shock proteins at the elevated temperature. While extracellular calcium is apparently not involved in the activation of the heat-shock protein synthesis, its removal from the culture medium has a modest stimulative effect on the synthesis of the glucose-regulated proteins. Our results are consistent with the hypothesis that the expression of these two sets of proteins are regulated by separate control mechanisms.     

Regulation of the synthesis of heat-shock proteins in heat-resistant variants of Chinese hamster fibroblasts

The synthesis of the major heat-shock proteins (hsp) was compared in normal and heat-resistant Chinese hamster fibroblasts which express higher levels of the 70 kDa heat-shock protein (hsp70). Following exposure to a variety of experimental conditions that induce the elevated synthesis of the hsp, higher relative levels of hsp70 and lower relative levels of hsp89 and hsp110 were found in the heat-resistant variants. This effect was observed with all inducers tested. The relatively greater synthesis of hsp70 and relatively lower synthesis of hsp89 occurred at all temperatures tested and was found to be independent of cell culture conditions. The relatively greater increase in the levels of hsp70 in the heat-resistant variants after a mild heat shock was found to be a reflection of elevated levels of messenger RNA coding for this polypeptide. These results indicate that the heat-shock response in mammalian cells displays coordinate regulatory features and that the alteration of the expression of one of the hsp may affect the expression of the others.     

Angiotensinogen production by rat hepatoma cells in culture and analysis of its regulation by techniques of somatic cell genetics

Angiotensinogen was synthesized by cells derived from the Reuber H35 rat hepatoma. Independent clones produced similar amounts of angiotensinogen, which corresponded to about four times more than expected for normal hepatocytes. The protein was secreted rapidly but could be visualized within cells using immunofluorescence. For one clone, it is shown that maximal angiotensinogen synthesis occurred during mid-exponential growth. Somatic cell genetics techniques have been used to investigate the regulation of angiotensinogen expression. Eleven clones of dedifferentiated variant hepatoma cells that failed to produce most or all of the liver specific proteins analyzed including albumin fell into two groups: Seven clones produced only 1-3% as much angiotensinogen as the differentiated clones, and four showed a reduction to 10-30%. Clones of the latter class were the only ones among the eleven analyzed that retained the potential to give rise to revertants, showing restoration of the differentiated state. All revertants fully restored angiotensinogen production, but only some of them re-expressed albumin. Somatic hybrids between differentiated hepatoma cells and one of the variants showed a substantial reduction in angiotensinogen production, whereas for some clones, albumin synthesis was fully maintained. These results show that regulation of the expression of angiotensinogen and of a second serum protein, albumin, was independent and that angiotensinogen synthesis was a faithful indicator of the general differentiation profile of all classes of clones.     

Oxidative stress induces a subset of heat shock proteins in rat hepatocytes and MH1C1 cells

Lipoperoxidative damage caused by exposure of isolated hepatocytes or cultivated hepatoma cells to ADP-iron or to 4-hydroxynonenal induces the synthesis of some proteins which are different under these two conditions but are always a subset of the proteins induced in each type of cells upon heat-shock (heat-shock proteins). For at least one of these proteins (hsp 31), induced by 4-hydroxynonenal, the increase is dose-dependent and the effect of heat and the chemical seems to be additive. Lipoperoxidation may be implicated in the induction of some of the heat shock proteins, but reproduces only incompletely the response of protein synthesis typical of heat-shock conditions.     

In Euglena gracilis, a heat-shock protein related to hsc73 is constitutive and stress inducible

Using monoclonal antibodies directed against different cytoplasmic isoforms of hsp70 proteins, namely, the constitutive hsc73 and the inducible hsp72 isoforms, we found that one isoform related to hsc73 was present in Euglena gracilis. This hsc73-like protein is expressed with a higher rate of synthesis in cells growing under heat shock than in control cells. Moreover, in cadmium-resistant cells, cultured at normal growth temperature, the rate of synthesis of this protein is constitutively increased. These results indicate that a heat-shock protein related to hsc73 is present in an ancestral eukaryote, Euglena gracilis, and that this protein may be constitutive and stress inducible as well.     

Proline dehydrogenase from Escherichia coli K12. Properties of the membrane-associated enzyme

The pattern of protein synthesis in hepatoma cell clones was analysed by two-dimensional separation of [35S]methionine-labelled proteins. The clones were derived from the differentiated Reuber H 35 hepatoma and showed differences in the expression of a number of liver-specific functions and the resistance to the growth-inhibitory effect of glucocorticoids. Five protein spots were observed in the extracts of the differentiated Faza 967 cells that were absent from the electrophoretogram of the dedifferentiated H 56 cells. This clone, on the other hand, displayed six spots absent from Faza 967 cells. The growth of both Faza 967 and H 56 cells was strongly inhibited by 1 microM dexamethasone. The dexamethasone-resistant clone 2, a dedifferentiated derivative of Faza 967 cells, synthesized two polypeptides that were not present in Faza 967 or H 56 cells and produced four polypeptides at a lower level than Faza 967 cells. The examination of the short-term effect of dexamethasone on protein synthesis in Faza 967 cells revealed nine induced and one repressed protein spots, which appeared to be in good agreement with earlier published data. It is concluded that dedifferentiation, although bringing about marked changes in certain liver-specific functions, such as enzyme activities or protein secretion, affects only a relatively small fraction of the genes expressed.     

Heat shock protein levels are not elevated in heat-resistant B16 melanoma cells

Heat-resistant variants have been selected from B16 melanoma cells and from surface mutants previously derived from them. The aim of the present study was to explore the possible role of heat shock proteins in the manifestation of this heat resistance. The major heat shock proteins evident after heating have subunit molecular weights of 68, 70, 89, and 110K on sodium dodecyl sulfate-polyacrylamide gels. The 68-kDa protein is not evident in any of the unheated B16 cell lines while the levels of the other heat shock proteins are elevated after heating. The constitutive levels of the 70, 89, and 110-kDa heat shock proteins were assessed after gel electrophoretic separation of proteins in several of the heat-resistant variants. No major differences were found in the levels of these proteins between the heat-sensitive parent lines and the heat-resistant variants. We therefore conclude that heat shock proteins are not a determining factor in the heat-resistant phenotype of B16 melanoma cells.     

Dedifferentiated variants of a rat hepatoma: analysis by cell hybridization

Two independent dedifferentiated variants, H5 and FaoflC2, derived from the Reuber H35 hepatoma, produce trans-acting diffusible substances(s) that extinguish the expression of liver-specific proteins when hybridized with a well-differentiated cell line of the same origin (Fao and Fu5-5, respectively). H5 x Fao hybrids show total and stable extinction of four liver functions and clonal variability in the expression of three others. FaoflC2 x Fu5-5 hybrids are initially flat (like FaoflC2 cells), and die in glucose-free medium where survival requires expression of hepatic gluconeogenic enzymes, but then evolve to hepatoma-like and finally round morphology; these latter cells express all liver functions analyzed including the gluconeogenic enzymes. Two exceptional clones that remained flat long enough for complete analysis showed extinction of all hepatic functions not expressed by FaoflC2 cells. We conclude that this transitory extinction reflects the action and then loss of extinguishing factor(s) contributed by FaoflC2. When crossed with BW1-J mouse hepatoma cells. FaoflC2 causes stable extinction of mouse aldolase B. We propose that production of extinguishing factor(s) is the rule for dedifferentiated variants.     

The human heat-shock protein family. Expression of a novel heat-inducible HSP70 (HSP70B'') and isolation of its cDNA and genomic DNA.

The human heat-shock protein multigene family comprises several highly conserved proteins with structural and functional properties in common, but which vary in the extent of their inducibility in response to metabolic stress. We have isolated and characterized a novel human HSP70 cDNA, HSP70B' cDNA, and its corresponding gene sequence. HSP70B' cDNA hybrid-selected an mRNA encoding a more basic 70 kDa heat-shock protein that both the major stress-inducible HSP70 and constitutively expressed HSC70 heat-shock proteins, which in common with other heat-shock 70 kDa proteins bound ATP. The complete HSP70B' gene was sequenced and, like the major inducible HSP70 gene, is devoid of introns. The HSP70B' gene has 77% sequence similarity to the HSP70 gene and 70% similarity to HSC70 cDNA, with greatest sequence divergence towards the 3'-terminus. The HSP70B' gene represents a functional gene, as indicated by Northern-blot analysis with specific oligonucleotides, hybrid-selected translation with a specific 3' cDNA sequence and S1 nuclease protection experiments. In contrast with HSP70 mRNA, which is present at low concentrations in HeLa cells and readily induced by heat or CdCl2 treatment in both fibroblasts and HeLa cells, HSP70B' mRNA was induced only at higher temperature and showed no basal expression. The differences in patterns of induction may be due to the special features of the promoter region of the HSP70B' gene.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

THE FUNCTION OF HEAT-SHOCK PROTEINS IN STRESS TOLERANCE

We earlier demonstrated that hsp68 is deficiently induced upon stress in the glucocorticoid-resistant, dedifferentiated Reuber rat hepatoma clone 2 cells, but is strongly activated in the differentiated, glucocorticoid-sensitive Faza 967 cells from which clone 2 was derived. We used the two cell types to address the questions whether hsp68 is specifically involved in the development of thermotolerance and/or thermoresistance or drug resistance. Our experiments show that clone 2 cells were not protected from the killing effect of heat by pretreatment with sodium arsenite, whereas Faza 967 cells were. These results strongly suggest a role of hsp68 in the development of thermotolerance in hepatoma cells. Stable heat-resistant variants of clone 2 cells were also isolated, where an increased basal expression of several hsps was observed together with the (at least partial) restoration of the heat-inducibility of hsp68. These results suggest that several hsps are needed to protect the critical biological processes at high temperature. The heat-resistant hepatoma cells also became resistant to several anticancer drugs. The multidrug resistance of the hepatoma variants correlates with the overexpression of the plasma membrane P-glycoprotein. Our results showing that severely stressed hepatoma cells overexpressed the mdr gene(s) raise the possibility that the P-gp may participate in protection against environmental stress such as heat.     

Enhanced constitutive expression of the 27-kDa heat shock proteins in heat-resistant variants from Chinese hamster cells

Four heat-resistant variants were isolated after treatment of Chinese hamster lung cells with the mutagen ethyl methane sulfonate, followed by a single-step selection procedure consisting in a severe hyperthermic treatment of 4 h at 44 degrees C. The isolated clones had a stable resistant phenotype for at least 150 generations during which they showed a 5,000-fold increased survival to a 4-h treatment at 44 degrees C when compared to wild-type cells. Comparative two-dimensional electrophoretic analyses of proteins revealed that, like induced thermotolerant wild-type cells (i.e., cells induced to a transient physiological state of thermotolerance by a sublethal heat conditioning treatment administered 18 h before), the heat-resistant variants had, at normal temperature, an increased content of a heat-shock protein with Mr of 27,000 (HSP27). In three of the four heat-resistant variants, the increased content of HSP27 was correlated with a two-fold increase in the constitutive level of the mRNA encoding HSP27. Chinese hamster HSP27 is composed of three species that differ in their relative isoelectric point, among which the two most acidic forms are phosphoproteins. In both the heat-resistant variant and wild-type cells, heat shock induces a rapid enhancement of the phosphorylation of HSP27: maximal phosphorylation occurs within 10 min upon changing the incubation temperature from 35 degrees to 44 degrees C. A concomitant shift in silver-staining intensity is rapidly detectable between the three isoforms, which seems to indicate that the two phosphorylated species represent post-translational modifications of the more basic species. It is concluded that most likely the enhanced expression of HSP27 is linked to the resistant phenotype of the variants. The study provides supporting evidence that both the content and phosphorylation status of HSP27 are determining factors in the ability of cells to survive hyperthermic treatments.     

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).     

Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.     

Thermotolerance in cultured hepatoma cells: cell viability, cell morphology, protein synthesis, and heat-shock proteins

Heat treatment at 42 degrees C of cultured Reuber H35 rat hepatoma cells induced both a rapid decrease of the rate of protein synthesis and the rounding up of the cells. Reincubation at 37 degrees C resulted in a gradual flattening of the cells, resumption of protein synthesis, and the synthesis of heat-shock proteins. During the recovery period cells developed a resistance toward a treatment which otherwise should lead to heat-induced cell death. Thermotolerance measured in terms of cell survival was paralleled by thermal resistance of protein synthesis and the cellular ability to refrain from rounding up under heat stress.