R. A. Coss

Mammalian cells adapted to growth at pH 67 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.

Altered proton extrusion in cells adapted to growth at low extracellular pH

Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO‐10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl−/HCO−3 exchange inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification. J. Cell. Physiol. 173:397–405, 1997.

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.

Heat sensitization of G1 and S phase cells by procaine HCl. II: toxicity and probability of dividing following treatment

Extended studies of the procaine sensitization of synchronous populations of CHO cells heated in monolayers were performed to help explain the mechanism for this sensitization. Cells were treated with 43.0, 45.5 and 43.0 degrees C + procaine, clonal survival of cells was determined, and time-lapse cinematography was used to monitor the time-dependent morphological changes and division delay of the various populations for up to 72 h following treatment. Procaine sensitized G1 and S phase populations to killing by 43.0 degrees C with thermal enhancement ratios of 7.4 +/- 0.5 and 7.9 +/- 1.1 (mean +/- SE), respectively. The division delays (39.2 +/- 3.5 h) of G1 cells exposed to 45.5 or 43.0 degrees C +/- procaine were longer than those (21.9 +/- 2.3 h) of S phase cells treated to comparable surviving fractions (SF). In these experiments the SF of the G1 populations was 0.50 +/- 0.17 versus 0.33 +/- 0.08 for the S phase populations. G1 populations heated with 43.0 degrees C alone (SF, 0.34 +/- 0.05) had division delays of 19.4 +/- 2.4 h. Colony formation correlated well with both G1 and S phase cells that had regular divisions subsequent to treatments; the heat-induced death of G1 and S phase cells correlated with interphase lysis and irregular divisions (including bipolar divisions yielding micronucleated progeny). Deaths due to both categories of lesions are increased by treatment with procaine; however, death due to interphase lysis is enhanced more by procaine. In summary, procaine HCl sensitized G1 and S phase cells comparably to killing by 43.0 degrees C.

THERMOTOLERANCE AND INTRACELLULAR PH IN TWO CHINESE HAMSTER CELL LINES ADAPTED TO GROWTH AT LOW PH

As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low‐pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42°C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42° heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO−3. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells.

Inhibiting induction of heat shock proteins as a strategy to enhance cancer therapy

Cancer treatments that incorporate thermal therapy and some systemic therapies induce the production of heat shock or stress proteins. The induced heat shock proteins could lessen the effect of the therapy by inhibiting apoptotic signaling and by acting as molecular chaperones to prevent irreversible cellular damage. Strategies that prevent the induction of heat shock proteins would result in more apoptosis and necrosis, improving the cancer therapy. This paper briefly reviews cancer therapies that induce the stress response, and proposes strategies to reduce the stress response.

Hsp27 protects the cytoskeleton and nucleus from the effects of 42°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°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°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°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°C-induced perinuclear collapse and resistance to 42°C-induced aggregation of nuclear proteins, in addition to the reduction in heat cytotoxicity, upregulation of intracellular pH and upregulation of hsp27.

Acute extracellular acidification reduces intracellular pH, 42°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°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°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°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°C per unit decrease in pHi than cells grown at pH 7.3. Acute acidification abrogated the 42°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.

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.

Thermal sensitisation by lonidamine of human melanoma cells grown at low extracellular pH.

Abstract Purpose: This study tested the ability of lonidamine (LND), a clinically applicable inhibitor of monocarboxylate transporters (MCT), to thermally sensitise human melanoma cells cultured at a tumour-like extracellular pH (pHe) 6.7. Materials and methods: Human melanoma DB-1 cells cultured at pHe 6.7 and pHe 7.3 were exposed to 150 µM LND for 3 h, beginning 1 h prior to heating at 42 °C (2 h). Intracellular pH (pHi) was determined using 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) and whole spectrum analysis. Levels of heat shock proteins (HSPs) were determined by immunoblot analysis. Cell survival was determined by colony formation. Results: Treatment with LND at pHe 6.7 reduced pHi to 6.30 ± 0.21, reduced thermal induction of HSPs, and sensitised cells growing at pHe 6.7 to 42 °C. When LND was combined with an acute acidification from pHe 6.7 to pHe 6.5, pHi was reduced to 6.09 ± 0.26, and additional sensitisation was observed. LND had negligible effects on cells cultured at pH 7.3. Conclusions: The results show that LND can reduce pHi in human melanoma cells cultured at a tumour-like low pHe so that the 42 °C induction of HSPs are abrogated and the cells are sensitised to thermal therapy. Cells cultured at a normal tissue-like pHe 7.3 were not sensitised to 42 °C by LND. These findings support the strategy that human melanoma cells growing in an acidic environment can be sensitised to thermal therapy in vivo by exposure to an MCT inhibitor such as LND.