Biomedical subjects

G M Hahn

Publications and source records attributed to G M Hahn.

At least 19 recordsLinked to original sources

PubMed1999

Increase in radiation-induced HPRT gene mutation frequency after nonthermal exposure to nonionizing 60 Hz electromagnetic fields.

It is widely accepted that moderate levels of nonionizing electric or magnetic fields, for example 50/60 Hz magnetic fields of about 1 mT, are not mutagenic. However, it is not known whether such fields can enhance the action of known mutagens. To explore this question, a stringent experimental protocol, which included blinding and systematic negative controls, was implemented, minimizing the possibility of observer bias or experimental artifacts. As a model system, we chose to measure mutation frequencies induced by 2 Gy gamma rays in the redox-sensitive hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in Chinese hamster ovary cells. We tested whether a 12-h exposure to a 60 Hz sinusoidally oscillating magnetic-flux density (Brms = 0.7 mT) could affect the mutagenic effects of ionizing radiation on the HPRT gene locus. We determined that the magnetic-field exposure induced an approximate 1.8-fold increase in HPRT mutation frequency. Additional experiments at Brms = 0.23 and 0.47 mT revealed that the effect was reduced at lower flux densities. The field exposure did not enhance radiation-induced cytotoxicity or mutation frequencies in cells not exposed to ionizing radiation. These results suggest that moderate-strength, oscillating magnetic fields may act as an enhancer of mutagenesis in mammalian cells.

Animals↗

PubMed1995

Stable overexpression of human HSF-1 in murine cells suggests activation rather than expression of HSF-1 to be the key regulatory step in the heat shock gene expression.

Transcription of the heat shock genes is regulated by the activation of the heat shock transcription factor (HSF-1). After heat shock, HSF-1 forms oligomers and binds to the heat shock element (HSE), which consists of several repeats of NGAAN located in the promoter region of the heat shock genes. HSF-1 is then phosphorylated, leading to the enhanced transcription of the heat shock genes likely by transactivation. We have stably overexpressed the human heat shock transcription factor-1 (HSF-1) in murine cells to investigate whether the regulation of the expression of the heat shock genes may partly reside at the level of HSF-1 expression. Human HSF-1 cDNA was cloned into a retroviral vector (pvhhsf-1) and was overexpressed in a murine fibroblast cell line. The overexpressed human HSF-1 is found in both the cytoplasm and nucleus of control cells but is translocated into the nucleus upon heat shock. Electrophoretic mobility shift analysis suggests that the human HSF-1 has constitutive DNA binding ability and its DNA binding ability is increased upon heat shock. Cross-linking experiments indicate that the overexpressed human HSF-1 is mainly a monomer under control conditions and forms oligomers upon heat shock. Immunoblotting shows that the human HSF-1 is phosphorylated upon heat shock and its apparent molecular weight is shifted up by at least 10 kDa. In spite of both the DNA binding ability and phosphorylation, the overexpression of human HSF-1 does not increase the transcription of murine HSP-70 mRNA or increase the synthesis of other HSPs after heat shock beyond that observed in control untransfected cells. An exception is the enhanced synthesis of a 47-50 kDa protein after heat shock and an apparent lack of induction of one HSP-70 kDa species when the protein pattern is analyzed by isoelectric focusing. Interestingly, cells overexpressing human HSF-1 show a 4-fold increase in the basal expression of luciferase when the plasmids containing the human HSP-70 promoter ligated to the luciferase reporter gene are transiently expressed in these cells. Murine cells overexpressing human HSF-1 are more resistant to the cytotoxic effects of heat when compared to the control untransfected cells, but the kinetics of thermotolerance development and decay is similar between HSF-1 transfected and untransfected cells. In conclusion, human HSF-1 protein in murine fibroblasts is modified in a similar fashion as the endogenous mouse HSF-1 after heat shock. However, the overexpression of HSF-1 does not result in overproduction of heat shock proteins after heat shock, perhaps because these cells contain abundant amounts of endogenous HSF-1.

Animals↗

PubMed1994

Correlation of heat resistance and HSP-70A mRNA levels in human tumor cells measured by competitive quantitative polymerase chain reaction.

PURPOSE: Several HSP-70 genes have been cloned and sequenced in human cells. Among these genes, the HSP-70A mRNA and protein levels correlate best with the development and decay of thermotolerance and intrinsic thermal sensitivity. Leukemic and nonleukemic human tumor cells express low levels of the normally heat inducible HSP-70A mRNA in control nonheated cells. Using a competitive quantitative polymerase chain reaction, we have measured the levels of mRNA for this gene and have correlated it with both transient and intrinsic thermal sensitivity of tumor cells. Such studies were also extended to tumor samples obtained from patients. METHODS AND MATERIALS: In these studies, the plasmid phHSP-70 which contains the entire human HSP-70A gene was modified by the insertion of the T7 promoter at the 5'-end untranslated region as well as the insertion of a 23 bp synthetic linker at the BamH1 site in the promoter region of the HSP-70A gene. The PCR primers were located such that the amplified fragment contained the linker. Using the T7 polymerase, the HSP-70A mRNA was transcribed from this plasmid (phHSP-70L) in vitro. A known amount of HSP-70A mRNA was then added to the total RNA prepared from the cell samples or from the tumor tissues obtained from patients. Using the components of the PCR reaction plus known amounts of HSP-70A mRNA synthesized from phHSP-70L and unknown amounts of total cellular RNA, the samples were amplified and analysed on a denaturing acrylamide gel. The PCR products obtained from phHSP-70L were 23 bp larger than the PCR products obtained from the cell samples due to the addition of the synthetic linker to the HSP-70A gene in phHSP-70L and therefore, the two products could be easily distinguished from each other and quantitated. The alpha-32P-dCTP incorporated in each sample was quantitated by AMBIS Scanner. When the 32P-counts were equal in the known and the unknown samples, the amount of the HSP-70A mRNA was taken to be equal in the known and the unknown sample. RESULTS: The results show that HSP-70A mRNA levels can be used to predict the survival levels during the development and decay of thermotolerance. In nonleukemic human tumor cell lines, there are as much as 40-50-fold induction of HSP-70A mRNA levels during the peak of thermotolerance. In leukemic cell lines, however, HSP-70A mRNA levels are induced only by three-fold during the same time period. These differences between the levels of HSP-70A mRNA positively correlate with the amount of tolerance development in leukemic and nonleukemic tumor cells. HSP-70A mRNA levels also vary in different tumor cells under nonheated conditions and there is a positive correlation between HSP-70A mRNA levels in nonleukemic human tumor cells and the level of their intrinsic thermal resistance. CONCLUSION: HSP-70A mRNA levels can be used to predict the intrinsic thermal sensitivity of nonleukemic human tumor cells.

Base Sequence↗

PubMed1994

Hyperthermia induces doxorubicin release from long-circulating liposomes and enhances their anti-tumor efficacy.

PURPOSE: To examine the possibility that hyperthermia would accelerate drug release from long-circulating liposomes, and enhance their antitumor activity. METHODS AND MATERIALS: Liposomes were prepared by thin film hydration technique. Hyperthermia was induced by ultrasound apparatus and a water bath heating system. The antitumor efficacy of treatment against RIF-1 tumor in C3H mice was evaluated by the tumor growth delay assay. RESULTS: In vitro drug release experiments demonstrated that increase in temperature from 37 degrees C to 41 degrees C resulted in about a sixfold increase in doxorubicin (DOX) release in a 1-h period. Increasing the temperature to 43 degrees C, resulted in only a modest additional drug release. Drug uptake studies showed that local hyperthermic treatment immediately following the drug administration dramatically enhanced Stealth liposome-encapsulated doxorubicin (S-DOX) uptake by tumors, but did not do so for free DOX. At 42 degrees C and at a dose of 10 mg/kg, the accumulation of S-DOX was about 10-fold and 2.5-fold higher than that with free drug and S-DOX at 37 degrees C, respectively. The antitumor efficacy study confirmed our hypothesis that the addition of hyperthermia to the treatment of RIF-1 tumors with doxorubicin encapsulated in long-circulating liposomes would enhance antitumor effects. Two hyperthermia treatments given at 24-h intervals appeared to be the most promising method of combining heat and long-circulating liposomes. The increased antitumor activity was not accompanied by increased toxicity, as determined by the body weight of the mice. CONCLUSION: Local hyperthermic treatment is able to accelerate DOX release from long-circulating liposomes, increase tumor uptake, and enhance their antitumor efficacy. The combination of local hyperthermia and long-circulating liposomes appears to show considerable promise in the treatment of localized diseases.

Animals↗

PubMed1994

Altered regulation of heat shock gene expression in heat resistant mouse cells.

PURPOSE: The differences in the heat shock gene regulation between the RIF-1 cell line and its heat resistant derivative TR4 are further characterized. METHODS AND MATERIALS: In vitro gel retardation assays were used to assess the presence of activated heat shock transcription factor in the two cell lines. The levels of the heat-inducible HSP 70.1, the constitutive HSC 70, the germ line-specific HSP 70.2, and the HSP 28 mRNAs in both untreated and iso-heated RIF-1 and TR4 cells were determined using the polymerase chain reaction coupled with the reverse transcriptase reaction. Induction and decay of induced heat shock protein synthesis was measured by 35S-methionine labeling of proteins. RESULTS: Unheated TR4 cells display characteristics of heat shocked RIF-1 cells. TR4 cells have a constitutively activated heat shock transcription factor and elevated levels of the HSP 70.1, HSC 70, and the HSP 28 mRNAs. Upon an equal heat dose of 45 degrees C, 15 min, the TR4 cells exhibited a more rapid onset in heat shock mRNA and protein induction than did the RIF-1 cells. During the recovery from heat shock, the activated heat shock transcription factor and the induced HSP70 mRNAs decayed more slowly in the TR4 cells, although the protein synthesis pattern of the TR4 cells returned to control levels more rapidly following heat shock than did protein synthesis of the RIF-1 cells. CONCLUSION: Unheated TR4 cells are similar to heat shocked RIF-1 cells at the transcriptional level. Induced HSP70 expression is modulated by the severity of the heat treatment (or the degree of heat damage) perceived by the cells rather than by the absolute heat dose given. We propose that the unheated TR4 cells are locked into the "ON" state of the heat shock response.

Animals↗

PubMed1994

Inhibitors of tyrosine and Ser/Thr phosphatases modulate the heat shock response.

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA-1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of beta-galactosidase in an HA-1 cell line containing the heat shock promoter ligated to the beta-galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA-1 cells with 500 microM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished beta-galactosidase induction. Reduced HSP synthesis was further confirmed by SDS-PAGE and Western blot analysis using anti-HSP-70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.

Animals↗

PubMed1994

Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing.

Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell-cell contact plays in this phenomenon. The formation of cell-cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca2+ restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell-cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing.

Animals↗

PubMed1994

Modulation of potassium channels by protein tyrosine kinase inhibitors.

Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels.

Acetylcysteine↗

PubMed1994

Selective expression of heat shock genes during differentiation of human myeloid leukemic cells.

Several studies have indicated a role for heat shock proteins during development and differentiation. In these studies we have examined the patterns of activation of the HSP-70A, HSP-70B, HSP-70B' and HSP-28 mRNAs and proteins during the differentiation of immature human leukemic cells to more mature progenitors by several differentiation-inducing agents. K562 cells activate the mRNA for HSP-70A, HSP-70B' and HSP-28 genes in the presence of hemin or sodium butyrate as cells differentiate into late erythroblasts. K562 cells become progressively more resistant to killing by heat shock during their differentiation to late erythroblasts. Further, selective inhibition of HSP-70A by antisense oligonucleotides to reduce HSP-70 kDa accumulation results in consistent reduction of hemoglobin production by 25-30% in K562 cells exposed to hemin, HL-60 cells differentiate into mature macrophages within 3 days following addition of PMA. HSP-70A mRNA levels increase within the first 2 h of PMA treatment and remain elevated for up to 3 days during the cells' gradual differentiation into mature macrophages. PMA and sodium butyrate treatment also cause elevated levels of HSP-28 mRNA expression; this increase is barely detectable at 24 h but is considerable at 72 h when about 90% of HL-60 cells are differentiated into mature macrophages or monocytes. These studies show that HSP-70A, HSP-70B' and HSP-28 may have specific roles during the differentiation of blood cell progenitors into erythrocytes or macrophages. Further, differentiation alters the thermal sensitivity of leukemic cells.

Base Sequence↗

PubMed1994

Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels.

In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K+) currents, activated by ionizing radiation (Kuo et al., 1993), play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6 +/- 4.0 mutations per 100,000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5 +/- 1.5. This increase, however, is abolished if either K+ channel blocker, CsCl or BaCl2, is present for 2 h following irradiation of the cells. RbCl, chemically similar to CsCl but known not to block K+ channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsCl or BaCl2 had no effect on radiation-induced cell killing.

Animals↗

PubMed1993

Purified mammalian HSP-70 KDA activates phosphoprotein phosphatases in vitro.

We have examined protein phosphorylation in the presence of purified mammalian HSP-70 kDa using the phosphoproteins in the rabbit reticulocyte lysate system as a model. Purified HSP-70 added to the rabbit reticulocyte lysate decreased the general protein phosphorylation by 50-80% as measured by PAGE analysis of proteins labelled with gamma-(32P)-ATP. Reduction in protein phosphorylation was not due to the ATPase activity of HSP-70 as measured by thin layer chromatography. The reduction in protein phosphorylation was also not due to the reduced activities of the protein kinases. However, using (32P)-labelled phosphorylase-alpha as a substrate in the phosphatase assay system indicated increases in the activity of protein phosphatase 1(PP-1) and/or 2A (PP-2A) by 20-40% relative to control in the presence of increasing concentrations of HSP-70. Using a variety of activators and inhibitors of the two major protein phosphatases, PP-1 and PP-2A, we found that Mn2+ caused a similar pattern of dephosphorylation of proteins as measured by PAGE analysis. Both okadaic acid and microcystin, two protein phosphatase inhibitors, largely counteracted the HSP-70 effect as measured by gel electrophoresis or when (32P)-labelled phosphorylase-alpha was used as a substrate. We conclude that in this system HSP-70 activates specific protein phosphatases.

Animals↗

PubMed1993

Potassium-channel activation in response to low doses of gamma-irradiation involves reactive oxygen intermediates in nonexcitatory cells.

Active oxygen species are generated during pathophysiologic conditions such as inflammation and ionizing radiation exposure. We tested the hypothesis that an early cellular event in response to these species involves regulation of ion channels. We exposed cells to gamma-irradiation or treated them with hydrogen peroxide, xanthine/xanthine oxidase, or [3H]thymidine and then monitored channel activity by the technique of whole-cell voltage clamping. Recordings showed that both normal and tumor cells exhibit an increase in K+ currents after treatment with radiation, H2O2, and xanthine/xanthine oxidase but not with high specific activity [3H]thymidine, suggesting that the signal for K+ channel activation originates at the cell membrane. A single noncytotoxic dose of 10 cGy induced measurable levels of K+ currents, suggesting that the induction of currents regulates biochemical changes in response to stress. To test whether channel activity is sensitive to active oxygen species, we pretreated cells with N-acetyl-L-cysteine (NAC) to increase cellular pools of free radical scavengers before radiation. In NAC-pretreated cells, K+ channel activation by gamma-irradiation was abolished. It has previously been shown that protein kinase C (PKC) is activated by ionizing radiation and can regulate K+ channels in some cells. However, the effect of radiation on induction of K+ channel activity was independent of PKC, since cells chronically exposed to phorbol esters still produced K+ currents after radiation. These results suggest that an early cellular response to oxidative stress is the activation of K+ channels.

Acetylcysteine↗

PubMed1993

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549.

Active oxygen species are generated in cells during pathophysiologic conditions such as inflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We investigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1993

Experimental hepatic tumor necrosis. Comparison of spin-echo and pulsed magnetization transfer contrast magnetic resonance imaging.

RATIONALE AND OBJECTIVES: We compared the effectiveness of pulsed magnetization transfer contrast (MTC) magnetic resonance imaging (MRI) and spin-echo MRI in detecting tumor necrosis. METHODS: Adenocarcinoma cells were transplanted in the livers of 12 syngenic BDIX rats. To induce various degrees of tumor necrosis, the rats were randomly assigned to the following groups: 1) control; 2) localized hyperthermia; 3) intralesional cisplatin; and 4) hyperthermia plus intralesional cisplatin. At day 7 after treatment, the rats were imaged using a 1.5-T imager with 1) multiplanar gradient-recalled echo sequence (MPGR) 500/8/20 degrees with and without magnetization transfer contrast (MTC); 2) spin-echo 2500/20,80, and 3) spin-echo 300/20 pulse sequences. The rats were then sacrificed and pathologic specimens were prepared using MR images as guidance. T2 and ratios of signal intensity after saturation to signal intensity before saturation (Ms/Mo ratios) of the necrotic and granulation tissues and viable tumors were determined in 10 rats. RESULTS: Compared with standard MPGR images, MPGR images with MTC provided better contrast between the pathologic tissues and normal liver. However, T2 values were more useful than Ms/Mo ratios in distinguishing necrotic areas from viable tumor. The T2 values of coagulative necrosis and granulation tissue were significantly different from that of viable tumor. No significant difference between the Ms/Mo ratios of the different pathologic tissues and normal liver was found. CONCLUSION: Pulsed magnetization transfer contrast MRI was inferior to spin-echo MRI in distinguishing necrotic from viable tumors in rat livers using the pulse sequences described, and none of the sequences studied was thought to be reliable enough for this purpose.

Adenocarcinoma↗

PubMed1992

Lower heat shock factor activation and binding and faster rate of HSP-70A messenger RNA turnover in heat sensitive human leukemias.

Normal bone marrow progenitors and some leukemic cells develop only a limited amount of thermotolerance. Further, once developed, thermotolerance decays at a faster rate than that normally observed in cells of nonhemopoietic origin. Thermotolerance induction and maintenance correlates with reduced levels of expression of various M(r) 70,000 heat shock proteins (HSP-70) mRNAs after heat shock. We have now compared the accumulation of HSP-70 proteins in heat-shocked human leukemic cells KG-1, HL-60, and K562 to that in Ht1080, a colon carcinoma cell line. We have found reduced accumulation of HSP-70 proteins in all leukemic cells. The rate of decay of HSP-70A mRNA, measured following heat shock by using actinomycin D treatment to inhibit further RNA synthesis, was more rapid in KG-1 and HL-60 cells compared to Ht1080 cells. The half-life of HSP-70A mRNA was 2 h in KG-1 and HL-60 cells while in Ht1080 cells it was > 7 h. HSP-70A mRNA is known to decay with a half-life of 2 h in unheated cells; this is increased to > 7 h following heat shock. We therefore postulate that leukemic cells lack the mechanism to stabilize HSP-70A mRNA after heat shock. One postulated mechanism for HSP-70 mRNA decay rate is known to be due to the nucleotide sequences at the 3'-untranslated region. We examined the 3'-untranslated region in leukemic cells. No sequence variations, however, were observed at either the genomic or the complementary DNA levels between leukemic or nonleukemic tumor cells. Heat shock factor activation and binding by gel retardation assays showed that KG-1 and HL-60 cells had a reduced heat shock factor binding to the heat shock element when compared to K562 and Ht1080 cells. Furthermore, HSF-1 mRNA was found to be expressed at relatively lower levels in HL-60 cells when compared to Ht1080 or KG-1 cells. In conclusion, reduced HSP synthesis and accumulation of leukemic cells after heat shock correlates with the reduction in heat shock factor-heat shock element binding and a faster HSP-70A mRNA decay rate that is observed in these cells.

Base Sequence↗

PubMed1992

Activation of potassium channels: relationship to the heat shock response.

We examined the possibility that whole cell currents are involved and possibly trigger the response of mammalian cells to heat shock. Heat-sensitive cells from a radiation-induced fibrosarcoma (RIF-1) and heat-resistant variants (TR-4, TR-5) were heated at 45 degrees C for 3-30 min. We observed induction of voltage-dependent currents after heating in the heat-resistant cells. These currents decayed to nonmeasurable levels over a period of 6 h. In RIF-1 cells, however, voltage-dependent currents were detectable during heating only; these currents then decayed rapidly. Tetraethylammonium (TEA) cations blocked the currents; changing the concentration of extracellular K+ modified the current-voltage (I-V) relationship. These currents, therefore, resulted from the activation of voltage-dependent K+ channels. Addition of TEA during heating sensitized TR-4 cells to heat but had no effect on the heat response of the RIF-1 cells. Continuous exposure of the RIF-1 cells to 2% (vol/vol) dimethyl sulfoxide (DMSO) for 7 days induced the expression of additional functional, voltage-dependent K+ channels; these gave rise to currents that were measurable after heating. In parallel, these cells became heat resistant. Addition of TEA to DMSO-treated cells blocked channels and returned the heat response almost to the pre-DMSO levels. Our data show a correlation between heat resistance and expression of K+ channels. Because resistance to heat very likely relates to the heat shock response, our data suggest that activation of channels may be a very early event in initiation of the heat shock response.

Animals↗

PubMed1992

Thermotolerance in the absence of induced heat shock proteins in a murine lymphoma.

Three murine lymphoma cell lines, CH1, a B-cell lymphoma, and VL3 and RDM4, both T-cell lymphomas, were tested for their ability to induce heat shock protein synthesis and thermotolerance after heat shock. All three lines could develop thermotolerance, but the persistence of tolerance was less than can be measured in nonlymphoid cell lines. Analysis of protein synthesis patterns by one-dimensional gel electrophoresis suggested that only the VL3 cells were capable of the induction of heat shock proteins. After two-dimensional gel analysis, however, the induction of one heat shock protein was evident in RDM4 cells. No induced heat shock proteins could be detected in the CH1 cells. These data provide strong evidence that, while the induction of heat shock proteins may be sufficient for development of thermotolerance, they are not necessary and that another mechanism is available to cells.

Animals↗

PubMed1992

Ultrasound-enhanced effects of adriamycin against murine tumors.

Earlier studies from our and other laboratories have demonstrated that ultrasound (US) enhances the cytotoxicity in vitro of the antitumor agent Adriamycin (Adr) (Harrison et al. 1991; Loverock et al. 1990; Saad and Hahn 1987, 1989; Yang et al. 1991; Yumita et al. 1987, 1989). We have now tested the possibility that this additional cytotoxicity can be translated into antitumor activity in vivo. Mice, bearing either a fibrosarcoma (RIF-1) or a melanoma (B-16) on their thighs, were injected with a single dose of Adr (10-20 mg/kg). The tumors were then heated locally to 41 degrees -43 degrees C for 30 min, either by insonation with US or by immersion of the animals' limbs into hot water baths. Antitumor efficacy was scored two ways: by serial measurements of tumor volume to determine the time for the tumor to double in size, or by determining the X-ray dose required to sterilize 50% of the tumors (TCD50) after the Adr-hyperthermia treatment. Both assays gave similar results. Ultrasound-induced hyperthermia was substantially more effective in enhancing Adr activity than was hyperthermia induced by the water bath. The mean-doubling time was 13 days for tumors treated with the combination of Adr and US and 6 days for tumors heated with a water bath immediately after injection of Adr. The TCD50 was 21.2 +/- 0.8 Gy for the combination of US and Adr and 36.1 +/- 0.9 Gy for the water bath heating and Adr.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗