Acute extracellular acidification reduces intracellular pH, 42 degrees C-induction of heat shock proteins and clonal survival of human melanoma cells grown at pH 6.7.

Two human melanoma cell lines, SK-Mel-28 and DB-1, were used for in vitro studies of the mechanisms underlying heat resistance of human tumour cells adapted to growth in acidic environments. Adaptation to growth at low pH was characterized by resistance to 42 degrees C cytotoxicity and accompanied by an increase in endogenous levels of Hsp70 and/or Hsp27. Acute extracellular acidification to levels below pH 6.5 was required to sensitize the melanoma cells to 42 degrees C. Furthermore, cells grown at low pH were more resistant to sensitization by acute acidification than cells grown at pH 7.3. The intracellular pH (pHi) of cells grown at pH 6.7 was less than the pHi of cells grown at pH 7.3 both before and after acute acidification. A pHi threshold existed for melanoma cells growing at pH 7.3 below which they became sensitized to 42 degrees C. This pHi threshold differed between the SK-Mel-28 and DB-1 cells. In contrast, a pHi threshold for heat sensitization did not exist for cells growing at pH 6.7: any reduction in pHi before heating resulted in increased cell killing. Since cells grown at low pH lack a pHi threshold for heat sensitization, they are sensitized more to 42 degrees C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42 degrees C-induction of Hsp70 and Hsp27 in the melanoma cells. The pHi thresholds for abrogation of these HSPs are slightly higher than or comparable with the thresholds for cytoxicity for each cell line grown at pH 7.3, but abrogation occurred over a narrower range of pHi compared with cytotoxicity. Abrogation of heat-induced expression of these HSPs correlates with cytotoxicity in both cell lines with the exception of Hsp27 expression in SK-Mel-28 cells. In conclusion, strategies that reduce pHi in melanoma cells growing at low pH, such as in acidotic regions of tumours, could selectively sensitize them to hyperthermia because they lack a pHi threshold for heat sensitization.

Quercetin sensitizes cells in a tumour-like low pH environment to hyperthermia.

Quercetin has been shown to act as a hyperthermia sensitizer by inhibiting the synthesis of heat shock protein 70 (HSP70) in a variety of tumour cell lines. It is most effective under conditions of low pH. This study was designed to test the hypothesis that quercetin suppresses thermotolerance development in cells adapted to growth at low pH and renders them as responsive as acutely acidified cells to hyperthermia-induced cytotoxicity. Chinese hamster ovarian carcinoma cells (OvCa) were exposed to 42 degrees C hyperthermia and/or quercetin (50-200 mm) at their growth pH of either 7.3 or 6.7 or after acute acidification from 7.3 to 6.7. Thermotolerance development was measured by colony survival. HSP70 synthesis and total protein synthesis were measured by radioactive precursor pulse labelling techniques. Quercetin, in a concentration-dependent manner, reduced the rate of total protein synthesis and increased cytotoxicity equally after acute acidification to pH 6.7 or growth at pH 6.7 at 37 degrees C, and to a greater extent than it did in cells at pH 7.3. At 42 degrees C, 100 mm quercetin inhibited total protein synthesis, HSP70 synthesis and thermotolerance development to a similar extent in cells grown at pH 6.7 or acutely acidified to pH 6.7. In contrast, quercetin reduced but did not completely inhibit HSP70 synthesis and thermotolerance development in cells grown and heated at pH 7.3. These results support the hypothesis that quercetin can specifically reduce thermotolerance development in tumour cells adapted to growth at pHe 6.7 so that they respond similarly to acutely acidified cells. Since many tumours are adapted to growth at low pH and may resist a wide variety of therapeutic modalities, inhibition of thermotolerance expression by quercetin may not only enhance the response to hyperthermia but the response to commonly used therapies such as chemotherapy and radiation.

Acute extracellular acidification increases nuclear associated protein levels in human melanoma cells during 42 degrees C hyperthermia and enhances cell killing.

Acute acidification is being investigated as a strategy to sensitize human melanoma to 42 degrees C hyperthermia. The present study was conducted to determine the effect of hyperthermia and acute extracellular acidification on the nuclear associated protein (NAP) levels, heat shock protein (hsp) 70 and hsp27 content, and cell survival of human melanoma cells cultured at pH 7.3 or pH 6.7. It was observed that NAP levels increased slightly in both populations after 2 h of heating and then decreased to control levels with increasing time of heating at the growth pH. However, the NAP levels continued to increase in cells acutely acidified to pH 6.3 prior to and during heating. Hsp70 was induced to comparable levels in cells heated at their growth pH; however, the hsp27 levels were greater in cells cultured and heated at pH 6.7 than in cells cultured and heated at pH 7.3. Acute acidification to pH 6.3 prior to and during heating suppressed the 42 degrees C induction of hsp70 and hsp27 in both cell populations. The melanoma cells cultured and heated at pH 6.7 were more resistant to cell killing than cells cultured and heated at pH 7.3. Both populations were sensitized to cell killing by acute acidification to pH 6.3. The results suggest that hsps induced during 42 degrees C treatment associate with aggregating NAPs, enhancing their detergent solubility, and that abrogation of induced expression of hsps during heating at pH 6.3 contributes to increased levels of insoluble NAPS. In conclusion, acute extracellular acidification inhibits 42 degrees C induction of hsps, increases NAP levels, and decreases cell survival in DB-1 human melanoma cells.

Hsp27 protects the cytoskeleton and nucleus from the effects of 42 degrees C at pH 6.7 in CHO cells adapted to growth at pH 6.7.

CHO cells are normally sensitized to hyperthermia by acidic pH. However, CHO cells adapted to growth in pH 6.7 medium become less sensitive to heat killing at the reduced pH. The adapted cells maintain their ability to develop thermotolerance at pH 6.7 and their steady state intracellular pH is elevated. Furthermore, the small molecular weight stress chaperone, hsp27, is elevated in unheated cells maintained at pH 6.7. This report documents that the cytoskeletal and nuclear components of the low pH adapted CHO cells are resistant to 42 degrees C-induced collapse and protein accretion, respectively. Hyperthermia induced a perinuclear collapse of the microtubular cytoskeleton and an increase in the amount of insoluble protein associated with the nuclei and nuclear matrix fractions in the control cells heated at pH 7.3 or heated after acute acidification to pH 6.7. Protection from these effects was observed in the low pH adapted cells heated at pH 6.7. Hsp70 does not appear to play a dominant role in the response of the adapted cells to 42 degrees C. The induction of hsp70 during heating is abrogated by pH 6.7 in cells cultured at either pH 7.3 or pH 6.7. The resistance of the microtubular cytoskeleton to perinuclear collapse and the absence of protein aggregation in the nucleus during 42 degrees C may be due to the elevated levels of hsp27 both before heating and during the heat treatment. In summary, the phenotype of CHO cells adapted to growth at low pH includes resistance of the cytoskeleton to 42 degrees C-induced perinuclear collapse and resistance to 42 degrees C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.

Betulinic acid sensitization of low pH adapted human melanoma cells to hyperthermia.

Betulinic acid is a known inducer of apoptosis in human melanoma that is most effective under conditions of low pH. It was hypothesized that betulinic acid, in combination with acute acidification and/or hyperthermia, would induce higher levels of apoptosis and cytotoxicity in low pH-adapted human melanoma cells than in cells grown at pH 7.3. DB-1 human melanoma cells, adapted to a tumour-like growth pH of 6.7, were exposed to hyperthermia (2h at 42 degrees C) and/or betulinic acid (4-10 microg/ml) and compared with cells grown at a physiological pH of 7.3 or after acute acidification from pH 7.3-6.3 or pH 6.7-6.3. Betulinic acid induced higher levels of apoptosis and cytotoxicity in low pH-adapted cells than in cells grown at pH 7.3, as measured by the terminal deoxynucleotidyl transferase (TdT) DNA fragmentation assay (TUNEL), the MTS cell viability assay, and single cell survival. Acute acidification of low pH adapted cells rendered them more susceptible to betulinic acid-induced apoptosis and cytotoxicity. In the presence of hyperthermia at 42 degrees C for 2 h, cells grown at pH 7.3 were not sensitized to heat killing by betulinic acid, whereas cells grown at pH 7.3 and acutely acidified to pH 6.3, cells adapted to growth at pH 6.7 and cells adapted to growth at pH 6.7 and acutely acidified to pH 6.3 were all similarly sensitized to heat killing by betulinic acid, with survival values of 5, 9 and 2%, respectively. It is concluded that betulinic acid may be useful in potentiating the therapeutic efficacy of hyperthermia as a cytotoxic agent in acidotic areas of tumours with minimal effect in normal tissues growing at pH 7.3.

Variability in glucose transporter-1 levels and hexokinase activity in human melanoma.

Melanoma exhibits heterogeneous growth patterns and widely varying sensitivities to multiple treatment modalities. This variability may reflect intrinsic genetic differences in factors giving rise to altered metabolism. Glucose is the primary energy source of tumours, including melanoma, and glucose transporter isoform 1 (Glut-1) and hexokinase are key rate-limiting factors in glucose metabolism. The levels of Glut-1 and total hexokinase activity were measured in 31 melanoma biopsies to determine the extent of tumour-to-tumour variability in these parameters. Relative Glut-1 levels were determined by Western immunoblot analysis using human anti-Glut-1 rabbit polyclonal antibody, and hexokinase activity was measured in the same samples by an enzymatic assay monitoring the reduction in the oxidized form of nicotinamide adenine dinucleotide phosphate (NADP+) (in nmol NADP+ reduced/min per mg protein). All melanomas were from patients who had received no therapy prior to surgery. Immediately after excision, tumour biopsies were disaggregated to single cells by collagenase and DNase and frozen in liquid nitrogen. Thirty human melanomas exhibited a 22-fold variation in levels of Glut-1 and 29 exhibited a nine-fold variation in total cellular hexokinase activity. Glut-1 levels and hexokinase activity were not correlated with one another. The broad range in Glut-1 levels and hexokinase activity observed between melanomas suggests that these glycolytic rate-limiting parameters that influence the rate of glucose metabolism may contribute to the variability in melanoma response to treatment modalities.

Absence of Crabtree effect in human melanoma cells adapted to growth at low pH: reversal by respiratory inhibitors.

Because many tumors are acidic and hypoxic relative to normal tissues, glycolysis and oxygen consumption were investigated in early-passage human melanoma cells adapted to growth at pH 6.7. In the absence of glucose, the basal rate of oxygen consumption in low pH-adapted cells was 75% of that in cells grown at pH 7.3. The rate of lactic acid production in low pH-adapted cells was increased 4-fold by exposure to 16.7 mM glucose compared with a 10-fold increase in cells grown at pH 7.3. Furthermore, in low pH-adapted cells the rate of oxygen consumption was stimulated by the addition of glucose in contrast to the inhibition of oxygen consumption by elevated glucose in cells grown at pH 7.3 (i.e., the Crabtree effect). Both low pH-adapted cells and cells grown at pH 7.3 exposed to glucose plus 0.35 mM meta-iodo-benzylguanidine (MIBG), an inhibitor of mitochondrial respiration, had oxygen consumption reduced by approximately 60% and lactic acid production increased by approximately 65% relative to glucose alone. Although adaptation to growth at low pH was associated with a loss of the Crabtree effect and a higher ratio of oxygen consumption to lactic acid production, the rate of glycolysis was the same in both growth conditions in the presence of 0.1 mM dinitrophenol, an uncoupler of ATP synthesis. This indicates that the glycolytic capacity of low pH-adapted cells remains unchanged. Therefore, tumor acute acidification and oxygenation can be achieved by exposure to hyperglycemia combined with MIBG to improve therapeutic response.

Hyperthermia does not affect rejoining of DNA double-strand breaks in a cell-free assay.

PURPOSE: Heat radiosensitization is poorly understood but is believed to be caused by an inhibition in the repair of radiation-induced DNA lesions. This inhibition in DNA repair may be caused either by direct heat inactivation of repair enzymes, or by heat-induced protein denaturation that leads to their precipitation onto nuclear chromatin structures, generating a barrier that prevents repair enzymes from reaching the damage sites. MATERIAL AND METHODS: A previously described (Ganguly and Iliakis, Int J Radiat Biol 1995, 68, 447-457) cell-free assay was introduced to evaluate rejoining of radiation-induced DNA double-strand breaks (dsb) in heated (45.5 degrees C, 20 min) nuclei prepared from A549 cells, in reactions assembled with extracts of non-heated and non-irradiated HeLa cells. The assay allowed the functional evaluation of the effect of precipitated nuclear protein on dsb rejoining. By combining heated nuclei with extracts of non-heated cells the assay avoided complications that would otherwise arise when intact cells are studied, where both nuclear structures and repair factors are heated and therefore potentially altered. RESULTS: It was observed that exposure of A549 cells to 45.5 degrees C for 20 min caused a 50% increase in the relative protein content of isolated nuclei but had no effect on the in vitro rejoining of dsb. In agreement with earlier reports, a greatly reduced rate of dsb rejoining was observed either in intact A549 or HeLa cells after exposure to heat. CONCLUSIONS: The results indicate that an increased retention of proteins in heated nuclei is not necessarily associated with an inhibition of dsb rejoining. While the in vitro system may only reproduce certain aspects of the in vivo conditions, the results suggest that protein accretion as a mechanism of heat radiosensitization requires further testing using functional assays.

Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts.

The role of Ku80 in the repair of DNA double-strand breaks (DSBs) was examined in fibroblasts derived from a Ku80 knockout mouse model described by Nussenzweig et al. (Nature 382, 551-555, 1996). Primary fibroblasts from Ku80+/+ and Ku80-/- mice were immortalized by transfection with plasmids containing either the human MYC proto-oncogene or the Simian virus 40 (SV40) T antigen and were used to measure induction and rejoining of DSBs after exposure to ionizing radiation. The number of DSBs in the cells was quantified by either asymmetric field-inversion gel electrophoresis (AFIGE) or clamped homogeneous electrical-field gel electrophoresis (CHEF). The latter method was introduced for a more reliable quantification of repair even when DNA degradation occurs in a fraction of the irradiated cell population during the postirradiation incubation time. The results confirm that Ku80-deficient mouse fibroblasts are sensitive to ionizing radiation and demonstrate that the increased radiosensitivity may result from a deficiency in DSB rejoining. The results further indicate that unless techniques are employed that allow for distinction between DNA degradation and DNA repair, erroneous conclusions may be drawn regarding the potential of cells to repair DSBs.

Mammalian cells adapted to growth at pH 6.7 have elevated HSP27 levels and are resistant to cisplatin.

HSP27 levels are elevated in two Chinese hamster cell lines and in a human melanoma cell line adapted to growth at pH 6.7. The level of HSP72 is elevated in the melanoma cell line but not in the hamster cell lines adapted to growth at pH 6.7. HSC73 levels are not elevated in any of the adapted cell lines. Low pH adapted cells from all cell lines are resistant to cisplatin. It is proposed that elevated HSP27 levels in low pH-adapted cells may play a role in resistance to hyperthermia and resistance to cisplatin.

Bicarbonate-dependent proton extrusion in Chinese hamster ovary (CHO) cells adapted to growth at pH 6.7.

A CHO cell model is described for in vitro studies of the mechanisms underlying heat resistance in cells adapted to growth in acidic environments. Adaptation is defined as a loss of pH 6.7-induced sensitization to 42.0 degrees C cytotoxicity and it is accompanied with an elevation of steady-state intracellular pH (pHi). CHO cells cultured between 75 and 100 days at pH 6.7 became fully adapted (6.7G cells), and the adapted phenotype was maintained for at least 100 additional days of culture at pH 6.7. The surviving fraction (SF) of 6.7G cells heated (42.0 degrees C) at pH 6.7 was comparable with that of cells cultured at pH 7.3 (7.3G cells) and heated at pH 7.3, while the SF of 7.3G cells acutely acidified to pH 6.7 and heated was an order of magnitude less. Although this resistance of 6.7G cells to killing was observed at 42.0 degrees C, it was not observed at 43.0 and 45.0 degrees C. Both 6.7G and 7.3G cells were able to develop comparable levels of thermotolerance during 42.0 degrees C at their growth pHs. However, in agreement with the literature, development of thermotolerance was reduced in acutely acidified 7.3G cells. An acute acidification of only 0.2 pH unit from pH 6.7 to 6.5 also reduced the ability of 6.7G cells to develop thermotolerance during heating at 42.0 degrees C. The acquired 6.7G phenotype reverted to the 7.3G phenotype following 17 days of culture at pH 7.3. Amiloride (0.5 mM), an inhibitor of the Na+/H+ exchanger (NHE), did not sensitize 7.3G and 6.7G cells to 42.0 degrees at their growth pHs. However, sensitization was observed for acutely acidified 7.3G cells. This is consistent with the hypothesis that extracellular acute acidification causes a decrease in pHi, and that the recovery from that decrease is achieved in part by activation of the NHE. An elevation of steady-state pHi, measured by analysing intracellular BCECF excitation spectra, was documented in a suspension assay for cells grown at pH 6.7 for 180 days. The elevation was bicarbonate-dependent (negligible in the absence of HCO3-, +0.17 pH units in the presence of HCO3-). These results suggest that the altered regulation of pHi in CHO cells adapted to pHe 6.7 is maintained by bicarbonate-dependent processes.

Response of the microtubular cytoskeleton following hyperthermia as a prognostic indicator of survival of Chinese hamster ovary cells.

PURPOSE: The response of the microtubular (MT) cytoskeleton to hyperthermia was assessed as a prognostic indicator of cytotoxicity. METHODS AND MATERIALS: Heat-induced collapse and subsequent recovery of the MT system were compared with survival for both nonthermotolerant (NT) and thermotolerant (TT) G1 populations of Chinese hamster ovary (CHO) cells. The response of the MT system was monitored using immunofluorescence staining. The G1 populations of NT and TT cells were heated by submersion in 45.0 and 43.0 degrees C waterbaths. RESULTS: Heat-induced perinuclear collapse of the MT system did not correlate with survival for the NT and TT populations. However, recovery of the organization of the MT cytoskeleton was correlatable with survival. The regression line of survival plotted as a function of MT recovery is fit by: y = -0.43 + 1.03x, r2 = 0.95 (p < 0.0005). CONCLUSION: Restoration of the organization of the MT cytoskeleton following hyperthermia may be used as a prognostic indicator of survival of CHO cells heated in G1.

Cellular localization of the bcl-2 protein and response to glucocorticoid stress.

We performed immunoelectronmicroscopy, immunofluorescence and subcellular fractionation studies of insect cells (Spodopetra frugiperda or SF9) infected with recombinant baculovirus containing bcl-2 cDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in pre-B cells carrying a bcl-2 gene activated by t(14;18) chromosomal translocation. By immunogold electron microscopy, bcl-2 was localized at several intracellular sites including the nuclear membrane, endoplasmic reticulum, mitochondria and plasma membrane. Immunofluorescence studies revealed the presence of the bcl-2 product throughout the cytoplasm, whereas biochemical fractionation studies indicated a similar pattern to that observed on electron microscopy. Our investigation clearly indicates that the bcl-2 product is expressed at several intracellular sites. Studies were also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in the distribution of bcl-2 protein were observed, more aggregated patches of gold labelled bcl-2 particles were found under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization necessary to prevent apoptosis.

Recovery of nuclear matrix ultrastructure of interphase CHO cells after heat shock.

Heat shock induces changes in G1 CHO cell nuclear matrix (NM) ultrastructure that may be related to heat-induced nuclear protein accumulation (Wachsberger and Coss, 1993, J. Cell. Physiol., 155:615-634). The present study quantitates recovery of alterations in NM fine structure in CHO cells heated in G1 and compares structural recovery with recovery of bulk RNA synthesis and surviving fraction (SF). Morphology of NM preparations was quantified 30 min and 20 hr following heat shock by 1) measurement of the number of fiber anastomosing points per unit area per NM, and 2) measurement of the length of fibers between points of anastomoses within individual NMs. Architectural recovery was nearly complete within 20 hr in cells heated at 43 degrees C or 45 degrees C with SFs of 0.27 or greater. No recovery of architecture was observed in heated cells with SFs of approximately 0.01 or less. The residual damage to NMs was associated with RNA-containing fiber networks as determined by means of RNase gold labeling. Recovery from inhibition of RNA synthesis following heat shock was related to recovery of NM architecture. It is suggested that 1) repair of NM architecture does not require full recovery of bulk RNA synthesis, and 2) partial or complete irreversible collapse of the NM may be responsible, in part, for heat-induced, interphase cell death.

Alterations in nuclear matrix ultrastructure of G1 mammalian cells following heat shock: resinless section electron microscopy, biochemical, and immunofluorescence studies.

Heat shock is known to inhibit vital nuclear functions associated with DNA and RNA metabolism. It has been proposed that the reported heat-induced excess protein accumulation in the nuclear matrix (NM) fraction may alter NM sites crucial for DNA and RNA processing. To test this hypothesis, we examined the fine structure of the NM in synchronous populations of G1 Chinese hamster ovary cells before and after heating by using the technique of resinless section electron microscopy. Heat did induce morphological alterations in the NM. The NM of control cells contained a honeycomb-like arrangement of fibers after chromatin removal. Following heat shock, NMs appeared as more highly anastomosing networks of polymorphic fibers and an overall increase in electron density was observed. Residual nucleoli from heated NMs underwent alterations in distributions of electron density both internally and at their peripheries. The increase in electron density observed in heated NMs was accompanied by an increase in protein mass and a relatively smaller increase in RNA mass as indicated by parallel sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and isotopic labeling (protein/DNA and RNA) studies. Some excess protein accumulation could also be directly localized onto NM fibers by use of antibodies to heterogeneous ribonucleoprotein complex antigens. It is concluded that alterations of NM fine structure can reflect the heat-stressed state of the cell, may account for the heat-induced inhibition of nucleic acid metabolism, and may be useful as an indicator of physiological or pathological stress in general.

Effects of hyperthermia on the cytoskeleton and cell survival in G1 and S phase Chinese hamster ovary cells.

The effects of acute hyperthermia on three cytoskeletal systems (microtubules (MT), microfilaments (MF), and vimentin intermediate filaments (VIMF] were observed in G1 and S phase Chinese hamster ovary (CHO) 10B cells using immunofluorescence microscopy and compared to cell survival. A scoring system was devised to express the degree of cytoskeletal collapse induced by heat and the degree of recovery 20 h following heat treatments. A positive correlation was found between recovery from heat-induced cytoskeletal disruption and surviving fractions (SF) of cells heated in G1 but not with SF of cells heated in S phase. Recovery of MT arrays, for example, averaged 96.5%, 71.6% and 20.3% for heat doses of 5 min, 15 min and 25 min, 45 degrees C, respectively. The corresponding SF (means) were 0.92, 0.68 and 0.23, respectively. However, in S phase cells, where restoration of MT and VIMF patterns averaged 94.2%, 83.8% and 33.0% for heat doses of 5 min, 15 min and 25 min, 45 degrees C respectively, SF were 0.70, 0.09 and 0.02. These results suggest that heat-induced cytoskeletal alterations may play a role in the death of cells heated in G1, and that these alterations do not significantly influence death of cells heated in S phase. This work is in agreement with previous studies showing that cells heated in G1 or S phase appear to die by different mechanisms, and further emphasizes the need to use synchronous populations of cells in order to understand the mechanisms whereby cells die following hyperthermia.

Acrylamide sensitization of the heat response of the cytoskeleton and cytotoxicity in attaching and well-spread synchronous Chinese hamster ovary cells.

The vimentin intermediate filament (VIMF) network is more sensitive to heat-induced disruption than either the microtubule (MT) or microfilament (MF) cytoskeletal (CSK) arrays in G1 Chinese hamster ovary (CHO) cells (Coss and Wachsberger: Radiation Research, 1987). We therefore investigated the effect of the VIMF disruptive agent, acrylamide (Eckert: European Journal of Cell Biology 37:169-174, 1985), on the heat response of synchronous CHO cells. Cells, either in the process of spreading (G1 or S phase) or in the well-spread state (S phase), were exposed to a nontoxic concentration of 5 mM acrylamide, heated, and processed for immunofluorescence microscopy 30 min or 20 hr following the heat shock. Recovery from CSK disruption was related to cell survival. CHO cells, either in the process of spreading or in the well-spread state, were sensitized to heat-induced CSK disruption and cytotoxicity by acrylamide. Recovery from CSK disruption correlated with surviving fractions of cells treated in the G1 phase but not with surviving fractions of cells treated in the S phase and was independent of the degree of cell spreading. This correlation suggests that damage to CSK structures may contribute to the death of cells treated in G1 but not necessarily to the death of cells treated in S phase. The degree of acrylamide sensitization of heat-induced CSK disruption was greater for cells exposed to acrylamide prior to spreading than for well-spread cells. Furthermore, normal spreading of cells was prevented when they were plated into medium containing acrylamide, suggesting that acrylamide interferes with the initial stages of attachment and spreading of these cells. These observations are interpreted in relation to the possible role that VIMFs, together with cortical MFs, may play in mediating cell surface focal contacts in the initial stages of cell attachment and spreading.

Interaction between vertebrate skeletal and uterine muscle myosins and light meromyosins.

The specific contributions of this work may be summarized as follows: (a) No hybridization of uterine and skeletal myosin occurs at pH 6.0 although previous studies have shown that hybridization does occur at pH 6.5 (B. Kaminer et al. 1976. J. Mol. Biol. 100:379-386) or 7.0 (T. Pollard. 1975. J. Cell Biol. 67:93-104) (b) Hybridization of uterine and skeletal light meromyosins (LMM) occurs at pH 7.0 but not at pH 6.0, which is analogous to the hybridization of myosins. (c) In hybridized paracrystals there is a uniform distribution of both uterine and skeletal LMM molecules because all the paracrystals have only one axial repeat pattern. This makes it highly likely that in hybridized filaments the two myosins are also uniformly distributed throughout the filaments. (d) The 14-nm repeat of white bands observed in paracrystals of uterine LMM formed at pH 6.0, compared with the 14-nm repeat of dark bands observed with skeletal LMM under the same conditions, probably reflects differences in surface charge density along the different LMM molecules.