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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.

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.

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.

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.

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.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.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.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.

Cells which have been adapted to growth at low extracellular pH (pHe) typically develop both an upregulation of steady state intracellular pH (pHi) and an ability to develop thermotolerance to 42 degrees C hyperthermia. These properties were acquired at different times, however. Days were required at pHe = 6.70 for two cell lines to adapt to low pHe by the thermotolerance criterion, but both had elevated steady state pHi values after only 4 hours at pHe = 6.70. A better correlation with adaptation to low pHe (as defined by hyperthermia) was found with changes in proton extrusion and the rate of pHi recovery after cytosolic acidification.

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.

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. © 1996 Wiley-Liss, Inc.

(1996). The effects of hyperthermia on the cytoskeleton: a review. International Journal of Hyperthermia: Vol. 12, No. 2, pp. 173-196.

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.

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°C or 45°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. © 1994 Wiley-Liss, Inc.

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. © 1993 Wiley-Liss, Inc.

Chinese hamster ovary (CHO) cells maintained in vitro at pH 6.7 were used to model cells in the acidic environment of tumours. CHO cells grown at pH 6.7 develop thermotolerance during 42 degrees C heating at pH 6.7 and their cytoskeletal systems are resistant to 42 degrees C-induced perinuclear collapse. Hsp27 levels are elevated in cells grown at pH 6.7 and are further induced during 42 degrees C heating, while Hsp70 levels remain low or undetectable, suggesting that Hsp27 is responsible for some of the novel characteristics of these cells. An anti-sense oligonucleotide strategy was used to test the importance of Hsp27 by lowering heat-induced levels of the protein. The response of the microtubular cytoskeleton to heat was used as an endpoint to assess the effectiveness of the anti-sense strategy. Treatment with anti-sense oligonucleotides prevented the heat-induced increase of Hsp27 levels measured immediately following heat. Treatment with anti-sense oligonucleotides also sensitized the cytoskeleton of cells grown at low pH to heat-induced perinuclear collapse. However, cytoskeletal collapse was not evident in cells grown at pH 6.7 and treated with 4-nt mismatch oligonucleotides or in control cells maintained and heated at pH 6.7. The cytoskeleton collapsed around the nucleus in cells cultured and heated at pH 7.3. These results confirm that over-expression of Hsp27 confers heat protection to the microtubular cytoskeleton in CHO cells grown at low pH.

This study tests the hypothesis that lowering intracellular pH (pHi) in melanoma cells grown at low extracellular pH (pHe) selectively abrogates 42 degrees C-induced heat shock protein (HSP) expression and reduces survival. Cells were acidified by a combination of a 0.2-pH-unit decrease in pHe coupled with the lactate/H+ transport inhibitor alpha-cyano-4-hydroxy-cinnamic acid (CNCn). A mild acute extracellular acidification was used to mimic the acute extracellular acidification observed in tumors that can be induced in vivo by oral glucose administration. CNCn blocks the activity of H(+)-linked monocarboxylate transporters (MCTs), particularly MCT isoform 1 (MCT-1). This transporter removes lactic acid from cells and has a high activity in DB-1 melanoma cells grown at low pHe. The effect of extracellular acidification combined with CNCn on pHi was measured in cells grown at pHe 6.7 and pHe 7.3. Cells grown at pHe 6.7 serve as an in vitro model for cells in an acidic tumor microenvironment. When cells were grown at pHe 6.7 and incubated with CNCn at pHe 6.5, the pHi decreased from 6.9 to below 6.5, and the 42 degrees C induction of HSP70 and HSP27 was blocked. The abrogation of HSP induction correlated positively with decreased clonogenic survival. In contrast, when cells growing at pHe 7.3 were acidified by a 0.2-pH unit to pHe 7.1, the inhibitor had less effect on pHi, which remained above 7.0. Under these conditions, the 42 degrees C-induction of HSPs was not inhibited, and cytotoxicity was not enhanced. These results indicate that a significant decrease in the pHi of melanoma cells can selectively sensitize the cells to 42 degrees C hyperthermia, possibly through the inhibition of HSP expression. This strategy could result in a therapeutic gain, because normal tissues, existing at a pHe above 7.0, would not be sensitized.

Although senescence is a defining property of euploid mammalian cells, its physiologic basis remains obscure. Previously, cell kinetics properties of normal tissue cells have not been considered in models for senescence. We now provide evidence that senescence is in fact the natural consequence of normal in vivo somatic stem cell kinetics extended in culture. This concept of senescence is based on our discovery that cells engineered to conditionally express the well-recognized tumor suppressor protein and senescence factor, p53, exhibit asymmetric cell kinetics. In vivo, asymmetric cell kinetics are essential for maintenance of somatic stem cells; ex vivo, the same cell kinetics yield senescence as a simple kinetic endpoint. This new “asymmetric cell kinetics model” for senescence suggests novel strategies for the isolation and propagation of somatic tissue stem cells in culture., National Cancer Institute (U.S.) (Grant CA-58619), National Cancer Institute (U.S.) (Grant CA-56690), National Cancer Institute (U.S.) (Grant CA-06927), Pew Charitable Trusts (Pew Scholars Program in Biomedical Sciences), U. S. Healthcare, Inc.

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.

This study examines the differential heat sensitivity of rounded suspension cells versus flattened monolayer cells. G1 populations of two different Chinese hamster ovary lines were used to eliminate possible cell cycle artifacts. The cell populations were heated at 43 and 45 degrees C. In all cases, cells treated in suspension were less sensitive to heat killing than cells treated as monolayers.

The sensitization of asynchronous Chinese hamster ovary (CHO) cells to 43 degrees C by procaine, lidocaine, and tetracaine was examined, with the pH of the medium carefully controlled at approximately pH7 and 8. Thermal enhancement factors for 43 degrees C were calculated for the surviving fraction of 0.01. The thermal enhancement factors of the local anesthetics were increased at approximately pH8 by factors of 2-12, depending on the local anesthetic and its concentration. The concentration of the uncharged free base form of the local anesthetic in the culture medium correlated positively with the thermal enhancement factors of each local anesthetic and in a near linear fashion with the thermal enhancement factors for procaine. However, the concentration of the cationic form of the local anesthetics in the growth medium did not correlate with heat sensitization. We conclude that the ability of local anesthetics to sensitize cells to heat killing is dramatically influenced by the extracellular pH, with increased sensitization at the more basic pH's. Secondly, it is the extracellular concentration of the free base form of the local anesthetics that correlates with heat sensitization.

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.

A portable battery-powered vibrating motor (36) that can be clipped on any standard syringe (10) provides vibration to a needle (16) to be inserted into a patient to minimize discomfort. The clip (24) is releasably attached to the syringe (10), and a holder (28) connected to the clip (24) releasably receives the motor (36). This enables the motor (36) to be used on other units and enables the syringe (10) and the clip (24) to be sterilized, without the motor (10).

The effects of the methylxanthine, caffeine, on heat sensitization was investigated using Chinese hamster ovary (CHO) cells. Caffeine sensitized CHO cells to heat killing by reducing both the shoulder and the slope of the 44°C survival curve. Heating was performed in suspension by addition of cells to preheated spinner flasks containing caffeine. Changes in intracellular free calcium levels, [ Ca2+] i, were measured at 37°C using the luminescent probe aequorin. Caffeine (1-5 mM) induced a transient increase in [ Ca2+] i at 37°C. The transient increase in [ Ca2+] i was reduced 15-fold when 5 mM caffeine was added to aequorin-loaded cells suspended in ${\rm Ca}^{2+}\text{-}free$ Hanks' balanced salt solution. However, 5 mM caffeine sensitized the cells to the same extent when they were suspended in either ${\rm Ca}^{2+}\text{-}containing$ or ${\rm Ca}^{2+}\text{-}free$ Hanks' balanced salt solution. The mechanism of heat sensitization by caffeine is still unknown.

Cell division in vegetative filaments of the green alga Oedogonium cardiacum is presented as an experimental system. We report on how we have used this system to study the effects of isopropyl N-phenylcarbamate (IPC) on the mitotic apparatus and on the phycoplast, a planar array of cytokinetic microtubules. Polymerization of microtubules was prevented when filaments, synchronized by a light/dark regime and chilled (2°C) while in metaphase or just before phycoplast formation, were exposed to 5.5 x 10-4 M IPC and then returned to room temperature. Spindles reformed or phycoplasts formed when these filaments were transferred to growth medium free of IPC. However, the orientation of both microtubular systems was disturbed: the mitotic apparatus often contained three poles, frequently forming three daughter nuclei upon karyokinesis; the phycoplast was often stellate rather than planar, and it sometimes was displaced to the side of both daughter nuclei, resulting in a binucleate and an anucleate cell upon cytokinesis. Our results suggest that IPC (a) prevents the assembly of microtubules, (b) increases the number of functional polar bodies, and (c) affects the orientation of microtubules in O. cardiacum. High voltage (1,000 kV) electron microscopy of 0.5-µm thick sections allowed us to visualize the polar structures, which were not discernible in thin sections.

Spermiogenesis in the macandrous, filamentous green algaOedogonium cardiacum is described at the ultrastructural level. The formation of the flagellar apparatus is similar to that in zoosporogenesis. Centrioles, appearingde novo in extranuclear, electron dense material, proliferate into two rows around the nucleus. Rootlet templates concurrently form between adjacent centrioles. The two rows migrate to the side of the cell and expand into a ring configuration. The centrioles (more correctly, basal bodies) extrude flagella while rootlet microtubules extend from the rootlet templates. Other components of the flagellar apparatus, including the fibrous ring and striated fibers, also form at this time. The flagellar apparatus of a mature sperm cell resembles that of a zoospore except the components are fewer in number and some are less well developed. A heterogeneous population of Golgi bodies in differentiating spermatids either contribute vesicles to the dome of the sperm cell, or else package and secrete extracellular products which are postulated to play a role in the release of sperm from the sperm packet.

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.

Non-toxic concentrations of verapamil (0.05 and 0.075 mM) and diltiazem (0.10 and 0.25 mM) sensitize CHO cells to heat killing at 44 degrees C. These drugs sensitize by reducing both the shoulder and the slope of the exponential survival curve. Exposure to verapamil or diltiazem in conjunction with the heat sensitizer procaine HCl at 44 degrees C reduced cell survival below that observed for procaine plus heat. The mechanism by which verapamil and diltiazem sensitize CHO cells to heat killing is not understood.

The binding of [ 14 C]isopropyl N -phenyl carbamate (IPC) to microtubular protein isolated from chick brains, and the effect of isopropyl N -phenyl carbamate (IPC) on the in vitro reassembly of microtubules was investigated. While [ 14 C]colchicine binds to microtubular protein, [ 14 C]IPC does not. Concentrations from 1 × 10 −4 M to 1 × 10 −3 M IPC do not prevent in vitro repolymerization of microtubular protein. IPC (1 × 10 −4 M) does not affect the rate of reassembly of microtubules. We conclude that IPC does not exert its effect through an interaction with microtubular protein; we suggest that IPC probably interacts with microtubule organizing centers.

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.

The role of the nucleus in the formation and maintenance of the contractile vacuole in amebae was investigated using microsurgery. Amebae were cut in two, forming nucleated and enucleated portions that either contained or lacked a contractile vacuole. The total numbers of cells and the numbers containing a contractile vacuole in these different categories were determined at intervals after the operation. In both nucleates and enucleates that initially contained a contractile vacuole, the contractile vacuole was maintained for as long as the cell survived. Enucleated fragments that lacked a contractile vacuole did not regenerate one. In contrast, nucleated portions initially lacking a contractile vacuole regenerated a contractile vacuole, usually within 1 h. The results indicate that the nucleus is required for the formation of a new contractile vacuole but that the nucleus is not necessary for the maintenance of the contractile vacuole.

The phenomena of heat-induced G1 delay and thermal resistance were compared in synchronous populations of CHO cells. Mildly toxic induction doses of 5 min (Single cell survival, (SCS) = 0.90 +/- 0.06) and 10 min (SCS = 0.69 +/- 0.12) at 45 degrees C resulted in G1 delays of 4.3 and 11.3 h, respectively. Thermal resistance was tested (30 min, 45 degrees C) for up to 32-92 h following the induction dose. Thermal resistance did not start to decay prior to 26 h following the induction dose. These data confirm reports by R. R. Read, M. H. Fox, and J. S. Bedford [Radiat. Res. 98, 491-505 (1984)] and G. L. Rice, J. W. Gray, P. N. Dean, and W. C. Dewey [Cancer Res. 44, 2368-2376 (1984)] that acutely heated G1 populations of CHO cells progress into S phase without a concurrent loss of thermal resistance, using 45 degrees C induction doses even less toxic than used by other workers.

Microtubular proteins isolated from beef brains were x-irradiated (500 to 30,000 rad) in the assembled or unassembled state in virto, and the effects of radiation on the samples were examined. Samples were irradiated in a water bath at 0 (ice slurry), 22 or 24, and 37/sup 0/C. The assembly of microtubules was monitored by following changes in optical density at 340 nm and/or changes in specific viscosity, combined with electron microscopy of the samples at various stages of experimentation. Samples containing microtubles were 30 (22/sup 0/C) to 50% (37/sup 0/C) more sensitive to inactivation by irradiation than samples containing unassembled microtubular proteins. Doses between 500 and 2000 rad had little effect on the assembly competency of the microtubular proteins. Doses of 5000 to 30,000 rad reproducibility inhibited the assembly of microtubules. More than 80% of the observed inactivation of microtubular proteins by 30,000 rad can be accounted for by the oxidation of critical sulfhydryls.