Role of dietary polyamines in a phase III clinical trial of difluoromethylornithine (DFMO) and sulindac for prevention of sporadic colorectal adenomas.

BACKGROUND: The polyamine-inhibitory regimen difluoromethylornithine (DFMO)+sulindac has marked efficacy in preventing metachronous colorectal adenomas. Polyamines are synthesised endogenously and obtained from dietary sources. Here we investigate dietary polyamine intake and outcomes in the DFMO+sulindac colorectal adenoma prevention trial. METHODS: Dietary polyamine data were available for 188 of 267 patients completing the study. Total dietary polyamine content was derived by the sum of dietary putrescine, spermine and spermidine values and categorised into two groups: highest (>75-100%) vs the lower three quartiles (0-25, 25-50 and 50-75%). Baseline tissue polyamine concentration and ODC1 genotype were determined. Logistic regression models were used for risk estimation. RESULTS: A significant interaction was detected between dietary polyamine group and treatment with regard to adenoma recurrence (P=0.012). Significant metachronous adenoma risk reduction was observed after DFMO+sulindac treatment in dietary polyamine quartiles 1-3 (risk ratio (RR) 0.19; 95% confidence interval (CI) 0.08-0.42; P<0.0001) but not in quartile 4 (RR 1.51; 95% CI 0.53-4.29; P=0.44). However, a lower number of events in the placebo group within dietary quartile 4 confound the aforementioned risk estimates. CONCLUSION: These preliminary findings reveal complex relationships between diet and therapeutic prevention, and they support further clinical trial-based investigations where the dietary intervention itself is controlled.

Rationale for, and design of, a clinical trial targeting polyamine metabolism for colon cancer chemoprevention.

Polyamine metabolic genes are downstream targets of several genes commonly mutated in colon adenomas and cancers. Inhibitors of ornithine decarboxylase, such as difluoromethylornithine (DFMO), and agents that stimulate polyamine acetylation and export, such as non-steroidal anti-inflammatory drugs (NSAIDS), act at least additively to arrest growth in human cell models and suppress intestinal carcinogenesis in mice. These preclinical studies provided the rationale for colon cancer prevention trials in humans. A Phase IIb clinical study comparing the combination of DFMO and the NSAID sulindac versus placebo was conducted. Endpoints were colorectal tissue polyamine and prostaglandin E2 contents and overall toxicity to participants. Participants in the Phase IIb study served as a vanguard for a randomized, placebo-controlled prospective Phase III trial of the combination of DFMO and sulindac with the primary study endpoint the prevention of colon polyps. Seventy percent of participants will have completed the three years of treatment in December 2006.

Effects of cycloheximide on thermotolerance expression, heat shock protein synthesis, and heat shock protein mRNA accumulation in rat fibroblasts.

A single hyperthermic exposure can render cells transiently resistant to subsequent high temperature stresses. Treatment of rat embryonic fibroblasts with cycloheximide for 6 h after a 20-min interval at 45 degrees C inhibits protein synthesis, including heat shock protein (hsp) synthesis, and results in an accumulation of hsp 70 mRNA, but has no effect on subsequent survival responses to 45 degrees C hyperthermia. hsp 70 mRNA levels decreased within 1 h after removal of cycloheximide but then appeared to stabilize during the next 2 h (3 h after drug removal and 9 h after heat shock). hsp 70 mRNA accumulation could be further increased by a second heat shock at 45 degrees C for 20 min 6 h after the first hyperthermic exposure in cycloheximide-treated cells. Both normal protein and hsp synthesis appeared increased during the 6-h interval after hyperthermia in cultures which received two exposures to 45 degrees C for 20 min compared with those which received only one treatment. No increased hsp synthesis was observed in cultures treated with cycloheximide, even though hsp 70 mRNA levels appeared elevated. These data indicate that, although heat shock induces the accumulation of hsp 70 mRNA in both normal and thermotolerant cells, neither general protein synthesis nor hsp synthesis is required during the interval between two hyperthermic stresses for Rat-1 cells to express either thermotolerance (survival resistance) or resistance to heat shock-induced inhibition of protein synthesis.

Effect of heating on lethality due to hyperthermia and selected chemotherapeutic drugs.

Because the rate of heating alters various cellular events associated with exposure to hyperthermia (including the rate of cell death), effect of this parameter on the cytotoxic interaction between selected anticancer drugs and hyperthermia was studied. The drugs cisplatin, 1,3-bis(2-chloroethyl)-1-nitrosourea, and adriamycin at 42.4 degrees C and bleomycin and methotrexate at 43 degrees C were tested in Chinese hamster ovary (CHO) cells in vitro. Heating times ranged from less than 3 minutes (immediate exposure) to 3 hours. For all drugs tested, synergistic cytotoxicity was not significantly altered by heating to peak temperature over 30 minutes as compared with immediate exposure. When heating to peak temperatures was prolonged to 3 hours, however, cell killing was markedly reduced, although significant sensitization to the drug remained. This was true despite the fact that, in CHO cells, heating to 42.4 degrees C over 3 hours produced no cell killing for up to 6 hours at that temperature. These results suggested that the mechanism(s) responsible for induced thermotolerance are probably different from those that cause cellular resistance to the cytotoxic effects of the drugs tested. These results may also partially explain the relative lack of clinical success with whole-body hyperthermia and chemotherapeutic drugs an anticancer treatment, because heating to therapeutic temperatures often requires 2-3 hours.

Effect of alpha-difluoromethylornithine on rectal mucosal levels of polyamines in a randomized, double-blinded trial for colon cancer prevention.

BACKGROUND: Polyamines (e.g., putrescine, spermidine, and spermine) are required for optimal cell growth. Inhibition of polyamine synthesis suppresses carcinogen-induced epithelial cancers, including colon cancer, in animal models. In a short-term phase IIa trial, we determined that low doses of alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (an enzyme involved in polyamine synthesis), reduced the polyamine content of normal-appearing rectal mucosa of subjects with a prior history of resected colon polyps. In a follow-up study, we have attempted to determine the lowest dose of DFMO that can suppress the polyamine content of rectal mucosa over a course of 1 year with no or minimal side effects. METHODS: Participants were randomly assigned to daily oral treatment with a placebo or one of three doses (0.075, 0.20, or 0.40 g/m2) of DFMO. Baseline and serial determinations of polyamine levels in rectal mucosa and extensive symptom monitoring (including audiometric measurements, since DFMO causes some reversible hearing loss at higher doses) were performed over a 15-month period. RESULTS: DFMO treatment reduced putrescine levels in a dose-dependent manner. Following 6 months of treatment, doses of 0.20 and 0.40 g/m2 per day reduced putrescine levels to approximately 34% and 10%, respectively, of those observed in the placebo group. Smaller decreases were seen in spermidine levels and spermidine:spermine ratios. Polyamine levels increased toward baseline values after discontinuation of DFMO. Although there were no statistically significant differences among the dose groups with respect to clinically important shifts in audiometric thresholds and nonaudiologic side effects, statistically significant higher dropout and discontinuation rates were observed in the highest dose group. CONCLUSIONS: Polyamine levels in rectal mucosa can be continuously suppressed by daily oral doses of DFMO that produce few or no side effects. A dose of 0.20 g/m2 can be used safely in combination phase IIb or single-agent phase III chemoprevention trials.

Dose de-escalation chemoprevention trial of alpha-difluoromethylornithine in patients with colon polyps.

BACKGROUND: alpha-Difluoromethylornithine (DFMO) is a potent inhibitor of carcinogenesis in experimental animal models. In these animal models, DFMO has been especially active in preventing carcinogen-induced epithelial cancers, including those of the skin, colon, breast, and urinary bladder. Although DFMO is known to exert its diverse biological effects by suppressing intracellular pools of the polyamines putrescine and spermidine, the precise mechanism by which polyamine depletion, induced by DFMO, suppresses carcinogenesis is unknown. PURPOSE: The specific aim of our study was to determine the lowest dose of DFMO that would deplete target tissue (colorectal mucosa) levels of these polyamines in humans who had undergone prior removal of colon polyps while producing minimal toxic effects. METHODS: A dose de-escalation chemoprevention trial of DFMO was conducted in 111 patients (36 female and 75 male) who were in generally good health, aged 39-79, and who had undergone colonoscopy for surgical removal of an adenomatous colon polyp greater than 3 mm within 5 years prior to entering the study. Groups of patients (12-20 patients per group) were orally treated with single, daily doses of DFMO ranging from 3.0 to 0.1 g/m2 for 4 weeks (28 days). Prior to initiation of DFMO treatment and at the end of treatment, six colorectal biopsy specimens were collected from each patient, along with serum samples. All biopsies were performed between 9 AM and noon to avoid possible effects of diurnal variations in laboratory end points. Samples for analysis of plasma DFMO levels were also collected during this time period on the day after the last day of drug administration. RESULTS: DFMO caused a decrease in both putrescine content and the ratio of spermidine to spermine for all dose groups down to 0.25 g/m2. Both putrescine content and the ratio of spermidine to spermine and changes in these parameters as a function of DFMO treatment decreased as a function of donor age. None of the 30 patients receiving either 0.25 or 0.5 g/m2 experienced any clinical ototoxicity in this trial. CONCLUSIONS: DFMO is both safe and effective in reducing colorectal mucosal polyamine contents when it is administered orally to patients at doses as low as 0.25 g/m2 for 28 days. No ototoxicity was observed at doses up to twice this amount. IMPLICATIONS: If DFMO is also found to be effective in suppressing polyamine contents in other target tissues, it may be useful in preventing a wide range of human epithelial cancers, including those of the prostate and breast.

Sensitization of Chinese hamster ovary cells to heat shock by alpha-difluoromethylornithine.

When exposed to alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, Chinese hamster ovary cells become increasingly sensitive to the cytotoxic effects of elevated temperatures (D.J.M. Fuller and E.W. Gerner, Cancer Res., 42:5046-5049, 1982). This sensitization becomes marked at times greater than 24 h after drug removal, and by 48 h, polyamine-depleted cells that have been exposed to 43 degrees C for 90 min have clonogenic survival values more than two orders of magnitude lower than control populations. Dose response studies demonstrate that, when measured 36 h after removal of the drug, hyperthermic cytotoxicity is maximally potentiated by exposure to DFMO for times as short as 2 to 4 h. A drug concentration of 1 mM for 8 h also elicits maximal response. An additional 8-h drug treatment 24 h after the first fails to further reduce survival in response to heat shock, suggesting the effects of the first exposure are persistent. Intracellular putrescine pools are depleted by the drug within 8 h, and spermidine levels continue to decline for up to 50 h. Consistent with these observations, ornithine decarboxylase (EC 4.1.1.17) activity is found to be reduced for up to 48 h after drug removal. The concomitant depression of spermidine is reflected in the elevation of S-adenosylmethionine decarboxylase (EC 4.1.1.50), which is substrate limited. Putrescine and spermidine show no sign of reaccumulation until approximately 4 days after DFMO exposure. Exposure to exogenous putrescine reversed the sensitization to heat shock induced by DFMO. This effect is quite specific for putrescine (1.4-diaminobutane) and is not replicated by other diamine homologues ranging from 1.3-diaminopropane to 1.8-diaminooctane. Polyamine-depleted cells express thermotolerance with kinetics similar to control cells although overall survival levels are lower. These results suggest that the mechanism of induction and expression of thermotolerance is independent of the role of acid-soluble polyamine pools in cellular responses to heat shock.

Delayed sensitization to heat by inhibitors of polyamine-biosynthetic enzymes.

Exposure of Chinese hamster ovary cells to the enzyme inhibitors methylglyoxal bis(guanylhydrazone and alpha-difluoromethylornithine (DFMO) results in increased sensitivity to hyperthermia. While methylglyoxal bis(guanylhydrazone) demonstrates pronounced cytotoxicity at moderate extracellular concentrations, DFMO is tolerated well by this cell line at concentrations of up to 10 mM, as assayed by clonogenic survival after treatments at 37 degrees. An 8-hr preincubation at 37 degrees with either drug elicits increasing sensitivity to 43 degrees hyperthermia treatments with time after removal of the drug. In contrast to results obtained by heating in the presence of the drugs during the 8-hr exposure, DFMO acts as the more effective sensitizing agent for this delayed effect on progeny of DFMO-treated populations. This phenomenon seems to result from depletion of intracellular putrescine, because the effect can be at least partially recovered by providing the cells with an exogenous source of this diamine. The potential for in vivo heat sensitization by the non-toxic agent DFMO has yet to be investigated but may have intriguing clinical possibilities.

The relationship between polyamine accumulation and DNA replication in synchronized Chinese hamster ovary cells after heat shock.

The relationship of polyamine accumulation and semiconservative DNA replication was studied in synchronous Chinese hamster ovary cultures, progressing through the cell cycle either normally at 37 degrees or after hyperthermic exposure (43 degrees for 1 hr) during G1 or S phase. In control cultures, intracellular polyamine levels decreased as cells divided and then reaccumulated as cells exited G1 and proceeded through the S and G2 phases. Immediately after cultures were exposed to 43 degrees heat for 1 hr in G1 phase, intracellular levels of spermidine and spermine were reduced compared to controls. Coordinate with the depletion of the intracellular levels of these polyamines following exposure at 43 degrees, extracellular levels of spermidine and spermine were increased. The ratio of intracellular to extracellular amounts of both these polyamines changed from 1 to 1.5 to approximately 0.2 to 0.3 after hyperthermic exposure. These cultures exposed to 43 degrees heat during G1 initially showed depressed levels of replicated DNA, but near-control rates of DNA replication were atained in a temporally related manner with the reaccumulation of intracellular spermidine and spermine levels. When cultures were exposed to 43 degrees heat in S phase, intracellular amounts of spermidine and spermine were again reduced and increased extracellular levels of these polyamines were observed. In these S-phase-treated cultures, cells were able to continue replicating their DNA but at a much reduced rate compared to controls. These results and other show that: (a) exposure of cells at 43 degrees causes a depletion of intracellular levels of spermidine and spermine, suggesting that an immediate aspect of thermal damage is a membrane defect that markedly affects the transport of these molecules across cell membranes; (b) exposure of either G1- or S-phase cultures to 43 degrees heat caused a depression of bulk DNA-synthetic rates resulting in a prolongation of S phase, and (c) the intracellular reaccumulation of spermidine and spermine following exposure of G1 cells to a 43 degrees heat shock is temporally related to the recovery of near-normal DNA synthetic rates in these cells.

Modulation of etoposide cytotoxicity and DNA strand scission in L1210 and 8226 cells by polyamines.

The anticancer agent etoposide (VP-16) produces DNA strand scission in intact tumor cells or isolated nuclei. This activity may be mediated by topoisomerase II, an enzyme capable of producing double strand breaks in mammalian cells. Two established tumor cell lines were examined to see whether polyamines, which alter DNA conformation and topoisomerase II activities, affected the cytotoxicity, strand scission, and antitumor efficacy of VP-16. L1210 murine leukemia and 8226 human myeloma cells were treated with alpha-difluoromethylornithine (DFMO) to reduce intracellular polyamine levels via inhibition of ornithine decarboxylase. The polyamines putrescine and spermidine were markedly reduced by a 48-h incubation with 50 microM DFMO. This DFMO concentration did not inhibit colony formation in either cell line, but did reduce the growth rate of both cultures. In contrast, VP-16 produced a dose-dependent inhibition of colony formation. This was especially marked in the 8226 cell line. This correlated with DNA single strand breaks (SSBs) detected by the alkaline elution technique. When cells previously treated with DFMO were exposed to VP-16, a synergistic inhibition of colony formation (determined by isobologram analysis) was observed. However, VP-16-induced SSBs were only marginally increased by the DFMO pretreatment. When putrescine was combined concurrently with VP-16, both the in vitro cytotoxic effects and the number of DNA SSBs in L1210 cells were significantly reduced. These results demonstrate that putrescine inhibits VP-16-induced SSBs and commensurate cytotoxic effects, while DFMO, which depletes intracellular putrescine and partially reduces intracellular spermidine, acts to produce synergistic cytotoxic effects when combined with VP-16.

Heat shock proteins in thermotolerance and other cellular processes.

Heat shock proteins appear to be causatively involved in the acquisition of thermotolerance in prokaryotes but not in eukaryotes. Further, the enhanced synthesis of hsps may be necessary for some cellular responses to stress but not others. In prokaryotic cells the development of thermotolerance, as measured by cell survival, is dependent upon protein synthesis. However, in eukaryotes, enhanced hsp synthesis following an inducing stress and prior to a subsequent heat shock is neither necessary nor sufficient for the development of thermotolerance as measured by colony-forming assays. The enhanced expression of hsps may be required for some mammalian cellular stress responses, such as the ability to reform both actin microfilament bundles and nucleolar morphology. These latter two thermotolerant responses have not been correlated with colony-forming ability. Future work should address the relationships between these various physiological responses to stress and determine if hsps function in some repair mode with regard to colony formation responses. Evidence is accumulating that hsps or their cognates may function in growth and differentiation in some manner as yet to be fully explained. Recent studies indicate that genes controlling cell division in E. coli may be linked to those of several stress regulons, and it would not be surprising to find a similar relationship in eukaryotes. At this time, it is important that studies investigating the role of hsps in stress and other cellular responses such as growth and differentiation define the specific gene (including its regulatory sequences) that encodes the protein being investigated, in order to avoid apparently contradictory and confusing reports of hsps expression.

Inhibition of ionizing radiation recovery processes in polyamine-depleted Chinese hamster cells.

Polyamines are involved in many cellular processes, including DNA structure and function. Since DNA, or some DNA-containing structure, is known to be the target for cell killing induced by ionizing radiation and a number of chemotherapeutic agents, we investigated the effects of polyamine depletion on cytotoxic responses of Chinese hamster cells to X-irradiation. Colony forming ability after single, acute radiation exposures of cells growing under oxic conditions was minimally affected by endogenous putrescine and spermidine depletion, achieved after treatment with alpha-difluoromethylornithine. Survival of cells rendered hypoxic and then irradiated was unaffected by alpha-difluoromethylornithine treatment. However, cellular recovery processes were nearly completely suppressed in polyamine-depleted cells, including sublethal damage recovery, as evidenced by split-dose irradiations in log phase cultures, and potentially lethal damage recovery, observed when growth-inhibited cultures were allowed time to repair radiation damage prior to being plated for colony formation. Both these recovery processes were restored by exogenous putrescine treatment. Reaccumulation of intracellular spermidine content closely correlated with restoration of potentially lethal damage recovery. Depletion of putrescine and spermidine pools had little effect on either single or double strand DNA break production or rejoining. These data demonstrate that both sublethal and potentially lethal damage recovery are polyamine-dependent processes in Chinese hamster cells, and imply that the mechanisms by which hamster cells recovery from these types of radiation damage are unrelated to their ability to rejoin DNA strand breaks, at least during the first hour after irradiation. Finally, these results suggest that the depletion of tumor polyamine content may be an effective method of enhancing the sensitivity of human tumors to fractionated radiotherapy.

Ornithine decarboxylase and polyamine levels in columnar upper gastrointestinal mucosae in patients with Barrett's esophagus.

Ornithine decarboxylase (ODC) activity was elevated in the premalignant metaplastic columnar epithelium (mean activity, 0.13 unit/mg protein, N = 18 individual samples from 18 patients), compared to either adjacent gastric (mean activity, 0.02 unit/mg protein, N = 9) or small intestinal (mean activity, 0.02 unit/mg protein, N = 9) epithelium in patients with Barrett's esophagus. Enzyme activity ranged from 0 (less than detectable) to more than 0.5 unit/mg protein in the metaplastic tissue. However, neither putrescine, spermidine, spermine (as individual parameters), nor total polyamine contents were related to ODC activity in the individual patient biopsies. Spermidine/spermine ratios ranged from 0.38 to 2.18 and were also not related to enzyme activity in any apparent manner. Nevertheless, cell strains derived from the metaplastic tissue were growth inhibited by alpha-difluoromethylornithine, an enzyme-activated, suicide inhibitor of ODC. In two different cell strains derived from Barrett's epithelium, growth was affected with drug concentrations as low as 0.05 mM. While the mechanism responsible for the elevation in enzyme activity is unknown, the regulation of polyamine metabolism appears to be altered in this premalignant tissue. The growth inhibition of Barrett's epithelium-derived cell lines by ODC inhibitors suggests a potential role for these compounds in the treatment of this disease.

Correlation between amounts of cellular membrane components and sensitivity to hyperthermia in a variety of mammalian cell lines in culture.

The weight ratio of either cholesterol or phospholipid to protein contents in 7 different cell lines, growing exponentially at 37 degrees, correlates positively with increasing resistance of the cells to subsequent hyperthermic cell killing. The relative heat resistance of each cell line is derived from survival curves obtained when the different cell lines are exposed to 43 degrees. Cholesterol and phospholipid amounts in the particulate fraction correlate with survival sensitivity to 43 degrees when the values are expressed per mg protein but not when expressed per cell number. Also, cholesterol:phospholipid molar ratios and the amount of protein in the particulate fraction do not display linear correlations with sensitivity of the respective cell lines to 43 degrees-induced cell killing. The relative degree of fatty acid saturation at 37 degrees also is independent of whether cells show a higher degree of heat resistance. These data suggest that lipid (both cholesterol and phospholipid):protein weight ratios correlate with increasing resistance of cells to an elevation in temperature. The major implication of these data is that major membrane components can influence and perhaps predict cellular survival to hyperthermia.

Reversal of resistance to methotrexate by hyperthermia in Chinese hamster ovary cells.

Our Chinese hamster ovary cells are extremely resistant to methotrexate (MTX) (100% survival after 500 microgram/ml for 13 hr). However, exposure to 43 degrees (but not 41 degrees or 42 degrees) for 1 hr sensitizes the cells to MTX so that a 50% cell kill in excess of that due to hyperthermia occurs. Treatment of cells at 43 degrees increases net MTX uptake by about 30% at 30 min but causes a substantial reduction after 1 hr. This negative effect is greater in cells continually heated at 43 degrees than in those exposed for only 1 hr. Treatment at 43 degrees for 1 hr also markedly increases efflux of MTX out of cells over the that 2 hr. Dihydrofolate reductase activity was found to decrease to about 50% of control values by 4 to 5 hr after exposure to 43 degrees. The biological half-life of dihydrofolate reductase in Chinese hamster ovary cells was determined to be about 4.5 hr, indicating that hyperthermia-induced cessation of protein synthesis may explain both the decrease in dihydrofolate reductase activity and the sensitization to MTX observed with heat exposure. In scheduling experiments, lethality due to exposure to 43 degrees for 1 hr in conjunction with MTX was maximum when 1-hr drug exposure began just at the end of heat treatment.

Rate of heating as a determinant of hyperthermic cytotoxicity.

In Chinese hamster ovary cells and in normal and transformed rat embryonic fibroblasts, survival as a function of time at 42.4 degrees was dependent upon the rate of heating from 37 degrees to 42.4 degrees. Unexpectedly, the untransformed rat fibroblasts were more heat sensitive than were the transformed cells, and the protective effect of slow rates of heating upon survival at 42.4 degrees was also more pronounced in the normal cells than in the transformed cells. In Chinese hamster ovary cells, total cellular cholesterol content and cell volume were found to change significantly with time at 42.4 degrees when cells were heated immediately (37 to 42.4 degrees within 3 min) but did not vary significantly during 6 hr at 42.4 degrees in cells heated from 37 to 42.4 degrees over 3 hr. Chinese hamster ovary cells heated immediately to 42.4 degrees also showed a significant drop in the protein content of the particulate fraction with time at 42.4 degrees. In contrast, cells heated over 3 hr showed a significant increase in the protein content of the particulate fraction with time at 42.4 degrees. These data suggest that, if cells are heated to hyperthermic temperatures over sufficiently long intervals, mechanisms have time to develop which protect the cell membrane against changes associated with cell death in rapidly heated cells. The protective effect of slow rates of heating may partially explain the relative lack of success thus far observed with the use of whole-body hyperthermia in which heating from 37 degrees to 42 degrees often requires 2 to 3 hr.

A transient thermotolerant survival response produced by single thermal doses in HeLa cells.

Continuous exposure of HeLa cells in culture to elevated temperatures (41-45 degrees) results in cell killing which increases exponentially as the time at the elevated temperature increases linearly. When cells are returned to 37 degrees after an initial thermal dose, cellular sensitivity to subsequent hyperthermic doses is reduced. Cell inactivation rates for cultures previously treated with 44 degrees for either 0.5 or 1 hr followed by incubation at 37 degrees for 2 hr, showed D0's of 1.1 and 1.5 hr, respectively, for subsequent thermal treatments at 44 degrees. Cultures receiving no prior hyperthermic dose had a D0 of 0.5 hr for treatments at 44 degrees for up to 3.5 hr. The viable progeny of cells treated with 44 degrees for 1 hr, however, had the same sensitivity to thermal doses at 44 degrees as did previously unheated cells. These results and others demonstrate that (a) single thermal dose produce a state of thermotolerance in HeLa cells to subsequent hyperthermic doses; (b) the degree of thermotolerance produced is dependent on the magnitude (i.e., temperature and time at the elevated temperature) of the first thermal dose; (cy thermotolerance does not develop at the elevated temperature but requires a return of culture temperatures to 37 degrees; (d) cellular acquisition of thermal tolerance is dependent on cell metabolism, as demonstrated by an inhibition of the effect at 0 degrees; and (e) this effect is a transient phenomenon which is lost as cells divide following the first thermal dose.

Sulindac sulfone inhibits K-ras-dependent cyclooxygenase-2 expression in human colon cancer cells.

Both the sulfide and sulfone metabolites of sulindac, a nonsteroidal anti-inflammatory drug, display anticarcinogenic effects in experimental models. Sulindac sulfide inhibits cyclooxygenase (COX) enzyme activities and has been reported to suppress ras-dependent signaling. However, the mechanisms by which sulindac sulfone suppresses cancer growth are not as defined. We studied the effects of these sulindac metabolites in human colon cancer-derived Caco-2 cells that have been transfected with an activated K-ras oncogene. Stable transfected clones expressed high levels of COX-2 mRNA and protein, compared with parental cells. K-ras-transfected cells formed tumors more quickly when injected into severe combined immunodeficiency disease mice than parental cells, and this tumorigenesis was suppressed by treatment with sulindac. Sulindac sulfone inhibited COX-2 protein expression, which resulted in a decrease in prostaglandin synthase E2 production. Sulindac sulfide had little effect on COX-2 in this model, but did suppress prostaglandin synthase E2 production, presumably by inhibiting COX enzyme activity. These data indicate that the sulfide and sulfone derivatives of sulindac exert COX-dependent effects by distinct mechanisms.

Cyclic AMP levels and types I and II cyclic AMP-dependent protein kinase activity in synchronized cells and in quiescent cultures stimulated to proliferate.

Cyclic AMP as well as the specific activity of cyclic AMP-dependent protein kinase decreased from the first two hours after Chinese hamster ovary cells in plateau phase were stimulated to proliferate by tripsinization of confluent cultures and dilution in fresh media. From two to five hours after this stimulation, the cyclic AMP level and the specific activity of cyclic AMP-dependent protein kinase increased two-fold. There was a 40--50% increase in the degree of activation of cyclic AMP-dependent protein kinase during this same time interval. In plateau cultures prior to being stimulated to proliferate, type I cyclic AMP-dependent protein kinase was the predominant soluble form of these enzymes. At five hours after release from plateau, the predominant type of cyclic AMP-dependent protein kinase was type II. However, there was also a significant amount of type I present at this time. Types I and II cyclic AMP-dependent protein kinases were differentially detectable during the cell cycle of Chinese hamster ovary cells synchronized by mechanical selection of metaphase cells following colcemid treatment. During mitosis, type I kinase was predominant with only a small amount of type II activity detectable. The amount of activity of type I then progressively decreased as cells entered G1. During early G1, there was no detectable activity of type II kinase, but its activity increased from mid to late G1 and then decreased during the S phase. These data show a tight temporal relationship between the levels of cyclic AMP, the total cellular pool of type I and II cyclic AMP-dependent protein kinases, and the degree of activation of these kinases as cells traversed G1 toward S phase. These data suggest that the expression of each type of kinase may be important for the regulation of substrate phosphorylation during the cell cycle.

G1 specific increases in cyclic AMP levels and protein kinase activity in Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized by selective detachment of cells in mitosis. The adenosine 3':5'-cyclic monophosphate (cyclic AMP) intracellular concentrations and cyclic AMP-dependent protein kinase activities were measured as these cells traversed G1 phase and entered S phase. Protein kinase activity, assayed in the presence or absence of saturating exogenous cyclic AMP in the reaction mixture, was lowest in early G1 phase (2 h after mitosis), increased 2-fold (plus exogenous cyclic AMP in reaction mixture) or 3.5-fold (minus cyclic AMP in reaction mixture) to maximum values in mid to late G1 phase (4-5 h after mitosis), and then decreased as cells entered S phase. Intracellular cyclic AMP concentrations were minimal 1 h after mitosis, increased 5-fold to maximum levels at 4-6 after mitosis, and decreased as cells entered S phase. Similar to the fluctuations in intracellular cyclic AMP, the cyclic AMP-dependent protein kinase activity ratio increased more than 40% in late G1 or early S phase. Puromycin (either 10 mug/ml or 50 mug/ml) administered 1 h after mitosis inhibited cyclic AMP-dependent protein kinase activity up to 50% by 5 h after mitosis, while similar treatment (10 mug/ml) had no effect on the increase in cyclic AMP formation. These data demonstrate that: (1) total protein kinase activity changed during G1 phase and this increase was dependent on new protein synthesis; (2) the increased intracellular concentrations of cyclic AMP were not dependent on new protein synthesis; and (3) the activation of cyclic AMP-dependent protein kinase was temporally coordinated with increased intracellular concentration of cycli AMP as Chinese hamster ovary cells traversed G1 phase and entered S phase. These results suggest that cyclic AMP acts during G1 phase to regulate the activation of cyclic AMP-dependent protein kinase.