13-Hydroxyoctadecadienoic acid is the mitogenic signal for linoleic acid-dependent growth in rat hepatoma 7288CTC in vivo.

Growth of hepatoma 7288CTC in male Buffalo rats is directly dependent on uptake of linoleic acid (LA) from the arterial blood. One to 5% of the LA taken up is converted to 13-hydroxyoctadecadienoic acid (HODE), an agent that enhances epidermal growth factor-dependent mitogenesis. The role of 13-HODE in LA-dependent growth of solid tumors is not known. In this study, we examined LA uptake and 13-HODE formation on growth of tissue-isolated hepatoma 7288CTC in vivo and on [3H]thymidine incorporation and DNA content during perfusion in situ. Fatty acid uptake and metabolite release were determined from arteriovenous difference measurements. Tumor-bearing and blood donor rats were fed either LA-sufficient or -deficient diets. Hepatoma 7288CTC removed LA from the arterial blood and released 13-HODE [and a small amount of 13-ketooctadecadienoic acid (KODE)] into the venous blood both in vivo and during perfusion. Treatment with the lipoxygenase inhibitor nordihydroguaiaretic acid (10 microM) did not affect tumor LA uptake, but inhibited release of 13-HODE and 13-KODE in vivo and during perfusion, suppressed growth in vivo, and inhibited [3H]thymidine incorporation during perfusion. The addition of 13-HODE to the nordihydroguaiaretic acid-containing whole blood perfusate increased the rate of [3H]thymidine incorporation 10 times and nearly doubled tumor DNA content; the addition of 13-KODE or 9-HODE had no effect. 13-HODE and 13-KODE were not released from tumors growing in rats fed a LA-deficient diet, and the rates of tumor growth in vivo and [3H]thymidine incorporation during perfusion were decreased. The addition of 13-HODE to the LA-deficient blood perfusate promoted tumor 13-HODE uptake and a dose-dependent increase in [3H]thymidine incorporation and tumor DNA content. These results provide strong evidence that 13-HODE is the mitogenic signal responsible for LA-dependent growth in hepatoma 7288CTC in vivo.     

Stimulation of tumor growth in adult rats in vivo during acute streptozotocin-induced diabetes

The effects of acute diabetes mellitus on the growth of Morris hepatoma 7288CTC and Jensen sarcoma were studied in fed, young (less than 200 g), and adult (greater than 250 g) rats. Animals were matched for tumor size and growth; the rates of tumor growth were the same in fed, young and adult nondiabetic rats. Diabetes was induced by the i.v. injection of streptozotocin (65 mg/kg total body weight) into tumor-bearing rats and changes in arterial blood nutrient concentrations were compared to changes in the rates of tumor growth and DNA synthesis. In young rats acute diabetes did not increase the blood concentrations of the fat store-derived nutrients and did not increase the rate of tumor growth. In adult rats, however, acute diabetes raised the arterial blood free fatty acid, glycerol, triglyceride, and ketone body concentrations to high levels and increased the rate of tumor growth about three times over that observed in untreated rats. Progress curves for the mobilization of host fat stores and for incorporation of [methyl-3H]thymidine into tumor DNA during the onset of diabetes showed that these activities were closely correlated in adult rats. Both processes began to increase 2 to 4 h after streptozotocin treatment, reached an initial peak at 12 to 16 h, decreased to a low point at 18 to 20 h, and then increased again to the new steady state after 23 to 24 h. The results indicate that the rate of tumor growth in rats in vivo is limited by the availability of a substance(s) present in the hyperlipemic blood of adult diabetic rats. The tight relationship between host lipolysis and tumor growth suggests that the substance(s) is derived from host fat stores.     

Stimulation of tumor growth in adult rats in vivo during an acute fast

These experiments investigate an increase in tumor growth that occurs in adult rats in vivo during an acute fast. The effects of feeding, fasting, and underfeeding on the growth of Morris hepatomas 5123C and 7288CTC in Buffalo rats and of Walker carcinoma 256 and Jensen sarcoma in Sprague-Dawley rats were studied. Animals were matched for tumor size and growth during a period of ad libitum feeding preceding the fasting or underfeeding. Tumor growth was documented by increased size and incorporation of [methyl-3H]thymidine into tumor DNA. Fasting increased the rate of growth of the tumors 3 to 4 times over that measured in fed rats. This effect began during the first day of fasting and ended abruptly on refeeding. After refeeding tumor growth slowed to the rate in fed rats. Tumors from fed or fasted rats were not different in cellularity or dry weight/g wet weight. A positive growth response in the tumor required lipolysis and ketosis in the host. No stimulation was observed during an acute fast in either immature rats or in mature rats whose weights had been reduced by underfeeding. These animals have small fat stores and show no increase in arterial blood free fatty acid or ketone body concentrations during an acute fast. Finally, underfeeding of adult rats raised the blood concentrations of these nutrients to values that were intermediate between those in fasted and fed rats. Tumor growth rates in these rats were intermediate between those in fasted and fed rats. The results support the proposal that an increase in availability of free fatty acids and/or ketone bodies is the stimulus that increases the rate of tumor growth during an acute fast.     

Mechanism for the antitumor and anticachectic effects of n-3 fatty acids

Dietary intake of the n-6 fatty acid (FA) linoleic acid (LA) has a strong growth-promoting effect on many rodent tumors and human tumor xenografts grown in immunodeficient rodents. n-3 FAs such as alpha-linolenic and eicosapentaenoic acids (EPAs), which differ from LA and arachidonic acid, respectively, by only a single double bond in the n-3 position, are recognized cancer chemopreventive and anticachectic agents. Understanding how this seemingly small structural difference leads to such remarkable functional differences has been a challenge. In a previous study, we showed that LA uptake, [3H]thymidine incorporation into DNA, and total DNA content were decreased in tissue-isolated hepatoma 7288CTC perfused in situ with arterial blood containing alpha-linolenic acid, EPA, or docosahexaenoic acids. The Ki for the inhibition of LA uptake and [3H]thymidine incorporation by alpha-linolenic acid was 0.18 and 0.25 mM, respectively. Here we show that the addition of alpha-linolenic acid or EPA to arterial blood inhibits tumor FA uptake, including LA, and the subsequent conversion of LA to the mitogen 13-hydroxyoctadecadienoic acid (13-HODE) in vivo and during perfusion in situ. [3H]Thymidine incorporation during perfusion in situ was also inhibited. Addition of 13-HODE to the arterial blood reversed the inhibition of [3H]thymidine incorporation but had no effect on FA uptake. These two n-3 FAs also inhibited FA transport in inguinal fat pads in vivo and during perfusion in situ in fed (FA uptake) and fasted (FA release) rats. The effects of EPA and talinolenic acid on transport of saturated, monounsaturated, and n-6 polyunsaturated FAs in hepatoma 7288CTC and inguinal fat pads during perfusion in situ were reversed by the addition of forskolin (1 microM), pertussis toxin (0.5 microg/ml), or 8-bromo-cyclic AMP (10 microM) to the arterial blood. We conclude that the antitumor and anticachectic effects of n-3 FAs on hepatoma 7288CTC and inguinal fat pads in vivo result from an inhibition of FA transport. These inhibitions are mediated by a putative n-3 FA receptor via a Gi protein-coupled signal transduction pathway that decreases intracellular cyclic AMP. A specific decrease in LA uptake and its conversion to the mitogen 13-HODE causes the tumor growth inhibition.     

Effects of cyanate, thiocyanate, and amygdalin on metabolite uptake in normal and neoplastic tissues of the rat.

Thiocyanate was found to resemble cyanate in its inhibitory effects on [3H]thymidine incorporation and the uptake of [32P]phosphate and [3H]amino acids in transplanted tumors of the BUF rat. The capacity to inhibit metabolite uptake in hepatomas and a colon tumor under conditions in which uptake was unchanged or increased in host liver was concluded to be a common feature of the action of cyanate and thiocyanate. Inhibition of [32P]phosphate uptake and [3H]thymidine incorporation into DNA of tumors was also observed after treatment of rats with amygdalin. With this drug, however, the action on tumors and livers of host rats was similar.     

Biochemical and ultrastructural studies on estrogen-induced pituitary tumors in F344 rats.

The hormone function, the metabolism of nucleic acids, and the ultrastructure of estrogen-induced pituitary tumors and of normal glands were examined in male F344 rats. The tumors had a high capacity for prolactin (PRL) synthesis, and the plasma levels of PRL were elevated 65-fold to 100-fold in the tumor-bearing animals. Uridine uptake and phosphorylation to nucleotides, as well as uridine incorporation into total RNA, were similar in tumors and normal glands, whereas [3H]thymidine incorporation into DNA was double in the former group as compared to the latter. After a [3H]uridine pulse, labeled RNA turnover was different in tumors and normal glands. Electron microscopy of the tumors revealed hypertrophy, degranulation, and hyperplasia of cells producing PRL with proliferation of their ergastoplasm in whorls. Other pituitary cell types were reduced in number. It is suggested that the whorl configuration caused the high rate of protein and PRL synthesis as well as the changes in RNA metabolism displayed by the tumors.     

Mechanism of growth enhancement of 7,12-dimethylbenz[a]-anthracene-induced mammary tumors in rats given high polyunsaturated fat diet

The mechanism of growth enhancement of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors by high fat diet was investigated in 169 female Sprague-Dawley rats fed a semisynthetic diet containing 0, 8 or 40% corn oil by weight. The animals, most of which were intubated with 10 mg of DMBA twice during the 8th week of age, were fed an unsupplemented or fat-supplemented diet for 2 months from the 12th to the 21st week of age and finally killed during the 21st week of age. Compared to the rats fed a fat-free diet, a significantly increased yield of mammary adenocarcinoma with a shorter latency was observed in the rats fed a 40% corn oil diet. DNA synthesis of the biopsied mammary tumors more than 1 month after diet switch-over was estimated in terms of the rate of [3H]thymidine incorporation into the tissue. Significantly higher DNA synthesis of the mammary tumors biopsied from proestrous hosts was found, as compared with the tumors in similar hosts at other stages of the estrous cycle. The greatest enhancement of DNA synthesis in small and rapidly growing tumors was observed in the 40% corn oil diet group. Feeding with 40% corn oil diet always resulted in elevation of the weight percentage of linoleic acid in both simple lipids and phospholipids of the mammary tumors. These data indicate that the effects of dietary fat on mammary tumor growth might be mediated by the enhancement of tumor response to certain hormones, and that the enhanced responsiveness of tumors was associated with increased linoleic acid in tumor lipids.     

Selective effects of two chloromethyl ketones on amino acid and phosphate uptake in rat liver and tumors.

Injection of L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) at a level of 10 mg/100 g body weight inhibited the incorporation of 3H-labeled amino acids into protein in Morris hepatomas 7777and 9618A2. The degree of inhibition was similar in cytoplasmic proteins and in histone and nonhistone nuclear protein fractions. There was no inhibitory effect on 3H-labeled amino acid incorporation in the livers of the tumor-bearing rats. The inhibitory effect of N-tosyl-L-lysine chloromethyl ketone (TLCK) on incorporation of 3H-labeled amino acids was observed in both the slowly growing hepatoma 7787 and the rapidly growing hepatoma 7777. In hepatoma 7777, TLCK (2.5 mg/100 g body wt) exerted a greater inhibitory effect on incorporation when administered 60 minutes before [3H]leucine injection than when injected simultaneously. Studies on tissue uptake of amino acids, thymidine, and phosphate indicated that inhibitory effects of TPCK and TLCK on active transport may be a major factor in the action of these drugs on macromolecular synthesis. The inhibitory effects of TPCK and TLCK seen in transplanted hepatomas and a colon tumor were not generally seen in normal tissues of the tumor-bearing rats.     

Blood thymidine level and iododeoxyuridine incorporation and reutilization in DNA in mice given long-acting thymidine pellets.

A long-acting thymidine pellet consisting of 190 mg of cholesterol and 60 mg of thymidine has been developed for the study of thymidine metabolism and reutilization in vivo. Implantation of such a pellet s.c. in adult mice will maintain the blood plasma concentration of thymidine at levels between 40 and 8 X 10(-6) M, which are from 36 to 7 times those of normal mice, for periods up to 48 hr. During this period, in vivo uptake and reutilization of [125I]iododeoxyuridine, a thymidine analog, into intestinal and tumor DNA were almost completely suppressed. While iododeoxyuridine reutilization is not large in normal proliferative tissue even in the absence of pellet implants, reutilization of over 30% was measured in large, rapidly growing ascites tumors. The inhibition of iododeoxyuridine incorporation by elevated thymidine blood levels is directly proportional to serum concentration. This appears to be due to a thymidine pool in rapid equilibrium with blood thymidine. This pool is at least 10 times larger than the 4-nmole pool of extracellular thymidine.     

Blood nutrient concentrations and tumor growth in vivo in rats: relationships during the onset of an acute fast

The rate of tumor growth in vivo in adult rats (250- to 350-g total body weight) is stimulated during an acute fast. No tumor growth stimulation is observed in fasted immature rats (less than about 200-g total body weight). The different tumor growth responses in rats of these two age groups appear to depend on the increased availability to the tumor of nutrients from host fat stores in adult rats. Immature rats, which lack significant fat stores, show neither hyperlipemia nor ketosis during fasting. These experiments were performed to determine the relationship between blood fat store-derived nutrient concentrations and the onset of stimulated tumor growth in fasted adult rats. Animals were matched for tumor size and growth during a period of ad libitum feeding preceding the fast. Tumor growth was documented by increased size and incorporation of [methyl-3H]thymidine into tumor DNA. Mobilization of host fat stores leading to increased blood concentrations of free fatty acids, glycerol, ketone bodies, and triglycerides started about 7 h after food was removed and reached its maximum after about 15 h. Increased rates of tumor growth and incorporation of thymidine into tumor DNA correlated closely with the higher circulating nutrient concentrations. Both the nutrient concentrations and tumor growth were decreased by refeeding. These findings suggest that the availability of nutrients derived from host fat stores may be rate limiting for tumor growth in vivo.     

Influence of mitoxantrone on nucleolar function in MDA-MB-231 human breast tumor cell line

The effect of mitoxantrone (DHAQ) on [3H]thymidine and [3H]uridine incorporation by exponentially growing MDA-MB-231, a human breast tumor cell line has been studied. The results have indicated that DHAQ was more effective in inhibiting [3H]thymidine than [3H]uridine incorporation in a concentration dependent manner. Following drug treatment at 20 ng/ml concentration, 50% inhibition of growth and [3H]thymidine incorporation were noted, whereas [3H]uridine incorporation was only inhibited by about 12%. At 2000 ng/ml of DHAQ the inhibition of cell growth, [3H]thymidine and [3H]uridine incorporations were 78%, 95% and 62%, respectively. Nuclear-associated radioactivity detected at light and electron microscope autoradiographic levels after [3H]thymidine and [3H]uridine incorporations, into DHAQ treated cells indicated that DHAQ prevented the accumulation of radioactivity into the nuclei in a concentration dependent manner. These results gave further indication that mitoxantrone induced a definitive alteration of nuclear template activities, correlated with nuclear functional-structural relation and suggested that the nucleoli were the primary site of DHAQ action.     

Enhancement of methylbenzylnitrosamine-induced esophageal carcinogenesis in zinc-deficient rats: effects on incorporation of [3H]thymidine into DNA of esophageal epithelium and liver

[³H]Thymidine incorporation was measured in esophageal epitheliumand liver DNA of rats fed a control diet ad libitum, a zinc-deficientdiet or a control diet, pair fed to the food intake of zinc-deficientrats. Additional groups of rats on each diet were dosed i.g.(2.5 mg/kg body wt) one or six times with the esophageal carcinogenmethylbenzylnitrosamine (MBN). The zinc-deficient diet significantlyincreased [3H]thymidine incorporation into esophageal epitheliumDNA at 14 days and reached a maximum at 28 days which was approximatelythree times that of the control ad libitum diet rats. Pair feedingsignificantly decreased incorporation into the esophageal epitheliumDNA relative to the control ad libitum diet, by approximately60% after 14 days. None of the diets affected [3H]thymidineincorporation in liver DNA. [³H]Thymidine incorporation wassignificantly inhibited for 48–72 h in the esophagealepithelium and liver DNA of all dietary groups after a singlei.g. dose of MBN before returning to predos-ing levels. Theperiod of inhibition of [3H]thymidine incorporation was lengthenedin all groups after six doses of MBN; the greatest increase(10 days) being noted in the esophageal epithelium DNA of thezinc-deficient rats. This inhibition was followed by a significantincrease above predosing levels in the esophageal epitheliumbut not in the liver. The maximum increase occurred 7 days afterthe completion of MBN dosing in the control ad libitum group,at 14 days in the control pair-fed group and at 36 days in thezinc-deficient group. These findings suggest that the enhancementof MBN-induced esophageal tumors by zinc deficiency is due inpart to the increased proliferation of the target cells witha concomitant greater accessibility of the cellular DNA to thecarcinogen.     

The apparent inhibition of urothelial DNA synthesis in neonatal rats by dietary saccharin

[³H]Thymidine ([³H]TdR) incorporation into urothelial DNA of male neonatal rats was measured autoradiographically at birth and during the first 3 weeks of life. The rats were derived from control parents and those fed saccharin (1, 3, 5 and 7.5%) in the diet from before pregnancy. [³H]TdR incorporation was inhibited and there were more lightly labeled cells (compared with controls), in all the saccharin-exposed rats in a rough dose-dependent manner. The results, in comparison with controls, suggest that saccharin exposure in utero causes DNA damage in the neonatal urothelium manifesting as reduced thymidine incorporation and a greater proportion of lightly labeled cells.     

Thiol proteinase inhibitor in the ascitic fluid of sarcoma 180 tumor-bearing mice

A thiol proteinase inhibitor (TPI) has been purified from the ascitic fluid of Sarcoma 180 tumor-bearing mice. The molecular weight of the inhibitor was estimated to be 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the substance inhibited papain, cathepsins B and L, but not cathepsins H and D and trypsin. The inhibitor also liberated kinin upon treatment with trypsin or mouse glandular kallikrein, indicating that the inhibitor is a kininogen, and the kinin liberated upon trypsinization was identified as bradykinin. An immunoreactive TPI with a molecular weight indistinguishable from that of the ascites TPI was found in plasma of non-tumor-bearing mice as well as that of tumor bearers. Plasma levels of immunoreactivity were increased up to twice the normal levels in tumor bearers inoculated with Sarcoma 180 or 3LL tumor cells. Supplementation of the purified ascites TPI into Sarcoma 180 culture medium caused a significant suppression of cell growth as well as [3H]thymidine incorporation at a concentration below that normally present in plasma. In contrast, addition of ascites-TPI to cultured mouse embryonic cells caused enhancement of cell growth as well as [3H]thymidine incorporation. These results indicate that in mice responding to tumor growth, a TPI corresponding to a kininogen is induced which may regulate tumor growth by countering tumor-related proteolytic activity.     

The effect of N-nitrosodimethylamine and N-nitroso-N-benzylmethylamine on [3H]thymidine incorporation into the DNA of target and non-target tissues in the zinc-deficient rat

The influence of dimethylnitrosamine (NDMA), a liver carcinogen and nitrosobenzylmethylamine (NBMA) as esophageal carcinogen on [3H]thymidine incorporation into DNA was studied in the esophagus, liver, forestomach and gastric-stomach of fasted zinc-deficient and pair-fed zinc-sufficient rats, measured 1 h after the thymidine injection. In the untreated animals, dietary zinc deficiency significantly depressed [3H]thymidine incorporation (89%) into the DNA of forestomach only. NDMA, administered 4 h before death at 30 mg/kg, produced 50-55% inhibition in [3H]thymidine incorporation in the esophagus of rats of both dietary groups. This inhibition became more pronounced in the forestomach, reaching 90-94% in the zinc-deficient forestomach and 63-86% in their zinc-sufficient counterparts at NDMA levels ranging from 5 to 20 mg/kg. NBMA at 2 mg/kg produced 60% inhibition in the DNA synthesis of zinc-deficient esophagus and 40% in the corresponding zinc-sufficient ones, this difference being significant at P less than 0.01. On the other hand, [3H]thymidine incorporation in the forestomach DNA was markedly lowered in the presence of NBMA. Recovery of DNA synthesis in the 4 tissues from a single dose of NDMA or NBMA was monitored up to 12 days. Following NDMA injection, [3H]thymidine incorporation in the forestomach of both dietary groups remained inhibited (3% of untreated control) for 5 days, a significant recovery (45% of untreated control) was observed only in the zinc-sufficient animals. Following NBMA injection, [3H]thymidine incorporation was also inhibited in the zinc-deficient esophagus for a longer time than in the zinc-sufficient ones. In autoradiographic studies, the percentage of cells showing 30 or more grains/nucleus was significantly decreased (P less than 0.001) in the NBMA-treated and marginally decreased (P less than 0.05) in the NDMA- or NBMA-treated zinc-deficient and zinc-sufficient rats as compared with the saline-treated zinc-sufficient controls. These results were discussed in the light of our previous findings that NBMA enhanced esophageal tumorigenesis in the zinc-deficient rats and that NDMA, a liver carcinogen produced forestomach tumors in the zinc-deficient but not in the zinc-sufficient rats.     

Estimation of radiation-induced interphase cell death in cultures of human tumor material and in cell lines

A short-term assay method able to estimate the radiation response of human cancer tissue samples would be of great advantage to the individualization of radiotherapy in cancer patients. However, the effect of radiation on [3H]thymidine incorporation by proliferating cells reflects a composite of cell cycle arrest and induced cell death pathways. Here we consider whether it is feasible to correct for cell cycle effects based on comparison of the effects of radiation and the mitotic inhibitor paclitaxel on [3H]thymidine incorporation. Sixty-two short-term (7-day) cultures of human tumor tissue from 61 patients with melanoma, gynecological cancer, brain cancer, and head and neck cancer, as well as 18 5-day cultures of low passage human tumor cell lines, were irradiated at doses from 2 to 9 Gy, or exposed to paclitaxel (200 nM). [3H]Thymidine incorporation was measured at the end of the incubation. Cell cycle times could be estimated from the paclitaxel data and were 2.7 to 18.6 days for melanomas, 2.5 to >40 days for carcinomas, 3.9 to 39 days for brain tumors, and 1.1 to 3.8 days for cell lines. The effects of radiation on [3H]thymidine incorporation varied widely (0-97% and 0-99% inhibition for 2 and 9 Gy, respectively), and in 23 of the clinical samples, but in none of the cell lines, radiation caused significantly greater inhibition of [3H]thymidine incorporation than paclitaxel (p < 0.05). We argue that that these differences reflect radiation-induced cell loss from G1 phase and/or S phase. Responses of short-term cultures of clinical tumor material to radiation, with appropriate correction for cell cycle effects, might have the potential to provide information on radiation-induced cell death in individual patients.     

Reversal of food restriction-induced inhibition of mouse skin tumor promotion by adrenalectomy.

Restricting the food intake of laboratory mice and rats markedly reduces the incidence of spontaneous and experimentally induced cancers. Using the two-stage skin tumorigenesis model in CD-1 mice, we report now that food restriction suppresses 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation of epidermal [3H]thymidine incorporation as well as TPA promotion of skin papillomas, whereas adrenalectomy completely reverses the inhibition in [3H]thymidine incorporation and tumor development. These results suggest that the adrenal gland may play an important role in mediating the tumor inhibitory effect of food restriction.     

Influence of lithium on mammary tumor growth in vivo

The possibility that lithium ions stimulate growth of mammary tumors in vivo has been suggested by their mitogenic action in vitro on normal and neoplastic mammary epithelium [8] and their clinical use as stimulators of neutrophil production in tumor-bearing patients treated with cytotoxic drugs [14,15]. Three experiments were performed to assess this possibility. Buffalo/N female rats received a single injection of N-nitrosomethylurea (NMU) at a dose known to produce mammary carcinomas in about 50% of animals under standard conditions. Under lithium treatment, the incidence of tumors did not increase significantly. Sprague-Dawley female rats treated with a single dose of 7,12-dimethylbenz[alpha] anthracene (DMBA), but showing no mammary tumors after 4 months, received lithium in their drinking water for 3 additional months. The number of late-appearing tumors was not increased by lithium treatment. Buffalo/N females with NMU-induced tumors were castrated, and the subsequent changes in tumor volume were compared in lithium-treated and control animals. The regression-regrowth curves were not altered by lithium treatment. These results are in contrast to the growth stimulatory capacity of lithium on mammary epithelium observed in vitro [8] and indicate it is very unlikely that lithium ions have an undesirable growth stimulatory action on primary mammary carcinomas in vivo.     

Differential sensitivity of normal and leukaemic haemopoietic cells to methionine deprivation by L-methioninase

The in vitro sensitivity of bone marrow cells from patients with leukaemia and from patients with non-malignant diseases to L-methionine removal by L-methioninase (L-methionine-alpha-deamino-gamma-mercaptomethane-lyase, EC 4.4.1.11) was determined using the incorporation of [methyl-3H]thymidine into acid-insoluble material as an index of survival. When compared with controls growing in medium containing 10 micrograms/ml of L-methionine, leukaemic cells showed a lower incorporation of [methyl-3H]thymidine after 24 h in the presence of 0.1 (normal 78 +/- 24%; leukaemic 26 +/- 18%, p less than 0.01) or 0.05 (normal 84 +/- 15%; leukaemic 50 +/- 21%, p less than 0.01) units of L-methioninase per ml. A similar differential sensitivity of leukaemic cells to L-methioninase was seen after 48 h of incubation. There was little effect on [methyl-3H]thymidine incorporation in the presence of boiled enzyme. Attempts to reverse L-methioninase toxicity with D-homocystine did not result in a differential effect on the normal cell population. The effects of L-methionine removal with L-methioninase were similar to those observed in L-methionine-depleted culture medium supplemented with 0.1 mM L-homocysteine. After 24 h in such depleted media leukaemic cells showed a lower incorporation of [methyl-3H]thymidine into acid-insoluble material (normal 88 +/- 17%; leukaemic 35 +/- 14%, p less than 0.01) and there was an elevation of the L-methionine-dependent enzymes: methionine adenosyltransferase, tRNA methyltransferase and S-adenosylmethionine decarboxylase. These results suggest the possibility of trying L-methioninase in the treatment of suitable leukaemias.     

In vitro modulation of cisplatin cytotoxicity by sparsomycin inhibition of protein synthesis

Inhibition of protein synthesis can alter cellular responsiveness to the classical anticancer drugs. The in vitro response of Chinese hamster ovary (CHO) cells to cisplatin with or without sparsomycin (Sm) was studied with the use of [3H]leucine and [methyl-3H]thymidine incorporation and clonogenic assay. Pretreatment of exponentially growing CHO cells with 1 microgram Sm/ml for 3 or 5 hours decreased [3H]leucine incorporation by 20% and resulted in significant resistance to cisplatin (P = .005). Sm in a concentration of 10 micrograms/ml reduced [3H]leucine and [methyl-3H]thymidine incorporation after 3 hours by 92 and 84%, respectively, and resulted in potentiation of the cisplatin cytotoxicity (P = .004). This effect was the same in the case of nonproliferating cells (P = .005), while protection due to Sm (1 microgram/ml) was seen only during cell proliferation. Simultaneous incubation and postincubation with Sm proved to have much less or no potentiating effect on cisplatin. The mechanisms of both protection and potentiation are still not clear, but our data indicate that Sm is a promising drug for further studies on the modulation of the cancer cell response to classical anticancer drugs.