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The folates present in liver, gut and tumour tissue were examined before and after autolysis. Before autolysis 10-formylfolate tetraglutamate (10-CHOFA(glu)4), 5-methyltetrahydrofolate triglutamate (5-CH3THF(glu)3) and possibly tetrahydrofolate polyglutamate(s) (THF(glu)n) were detected. Liver contained all 3 species whereas no 5-CH3THF(glu)3 was present in the tumours; gut showed an intermediate situation. After autolysis the predominant monoglutamates formed were 5-CH3THF in the liver, 10-formylfolates in the gut and possibly tetrahydrofolate (THF) in the tumour extracts. These differences illustrate changes in tissue folates with the proliferation rate of the tissue and suggest an explanation for the methionine auxotrophy of Walker 256 carcinosarcoma cells.

The hypothesis that there is prostaglandin-mediated hypercalcaemia associated with the Walker carcinosarcoma in the rat was tested by measuring PGE production during the development of the hypercalcaemia, and determining the effects of inhibition of prostaglandin synthesis on serum calcium concentration. Parathyroid hormone (PTH) activity was estimated by the determination of the serum concentration of immunoreactive PTH. There was a 3-fold increase in the urinary excretion of 7α-hydroxy-5,11-diketotetranor-prostane-1,16-dioic acid (PGE-M), a major urinary metabolite of the E prostaglandins from basal levels. Treatment with indomethacin, a potent inhibitor of prostaglandin synthesis, did not lower serum calcium concentrations with two different doses (1·6 mg/kg/day orally and 5 mg/kg/day i.m.); effective inhibition of prostaglandin synthesis was demonstrated by the suppression of PGE-M excretion rates below basal levels. Serum concentrations of immunoreactive PTH were not significantly altered by either tumour growth or indomethacin. Dexamethasone (0·5 mg/kg/day i.m.) attenuated both the increased urinary excretion of PGE-M and the rise in serum calcium concentration, suggesting that one or several lipoxygenase products might be the actual mediators of the hypercalcaemia. We conclude that the hypercalcaemia in the rat with Walker carcinosarcoma is probably not mediated by E-prostaglandins and probably not by any other product of the cyclo-oxygenase pathway. The increased PGE turnover may be considered as a biochemical marker of tumour load, but not as an indicator of a prostaglandin-mediated hypercalcaemia.

The Walker 256 carcinosarcoma was shown to lack the enzyme 3-ketoacid CoA transferase. This suggests that ketone bodies cannot be used as a major substrate for the energy metabolism of this tumour. Systemic ketosis (1-2 mM acetoacetate plus 3-hydroxybutyrate) was induced both in tumour-bearing and in non-tumour-bearing rats with a diet containing 70% medium chain triglyceride. However, in rats bearing the Walker 256 tumour, this dietary ketosis did not reduce the tumour growth rate nor did it prevent the subsequent decrease in host body weight. Host body nitrogen losses were similarly unaffected. The ketosis induced in tumour bearing rats was shown to be abnormal since the blood glucose concentration of ketotic, tumour-bearing rats was significantly higher compared with that of ketotic non-tumour bearing rats (5.2 +/- 0.4 mM cf 3.4 +/- 0.6 mM, P less than 0.01). These results may partly explain why systemic ketosis failed to alter the growth and cachexia induced by the Walker 256 carcinosarcoma.

Subdermal inoculation of the foot of the rat with lethally irradiated (LI) Walker tumour (W256) cells, mixed with viable (V) W256 cells, decreased the latent period for initiation of allogeneic tumour growth without significantly affecting its rate. This Révész effect decreased with increase in the number of inoculated V cells, and with decrease in age of recipient. LI cells of a different (Y-P388) rat tumour exerted a Révész effect, even in recipients which had been immunized with LI (Y-P388) tumour cells. Local pre-irradiation of the site of inoculation of V cells decreased both the latent period and rate of tumour growth. It acted independently of a Révész effect, and the decrease in tumour growth rate was partly due to emigration of V cells from the inoculum, producing metastases. LI, but not heat-killed cells, induced prolonged swelling of the tumour bed in unimmunized and tumour-immunized rats, which, unlike inflammatory swelling, was inhibited by pre-irradiation of the foot. It is postulated that the Révész effect is due to enhancement of survival of V cells by trophic substances which are principally elaborated by LI (AND V) cells, but also by the tumour bed, due to innate growth and trophic reactions of its tissues to the presence of tumour cells.

The metabolism of 2-[14C] folic acid has been studied in rats with an implanted Walker 256 tumour and in a closely matched group of controls. In animals with tumours, more of the labelled folic acid is converted to 10-formyltetrahydrofolate and 10-formylfolate than in normal animals. No 5-methyltetrahydrofolate could be detected in tumour tissue, or in the livers of tumour-bearing animals. When a mixture of 2-[14C]- and 3',5',9-[3H]-folic acid is given to tumour-bearing rats a similar pattern of metabolites is found. There is apparenly less scission of the folate molecule in tumour-bearing rats than in normal rats.

The 32P uptake in a mammary tumour induced by DMBA and in the Walker 256 carcinosarcoma was measured by external GM -tubes. The uptake was significantly higher than in the skin. During exposure to a synchronized light regime a circadian variation was present in the 32P uptake of the hormone-dependent DMBA-induced tumour. The maximal 32P uptake was in the dark period, in which the highest temperature in the tumour has also been found (Møoller and Bojsen, 1975). In the hormone-independent Walker 256 carcinosarcoma there was no periodicity in 32P uptake. No variation in 32P uptake was registered in the skin of normal controls or in tumour-bearing rats.

Polyclonal antibodies have been raised in rabbits to a haemocyanin adduct of a reductively-activated, fluorinated analogue of misonidazole. Fluorescence immunohistochemical studies show that the polyclonal antibodies bind to spheroid sections and tumour sections in patterns similar to those revealed by autoradiographic studies with a tritium-labelled derivative of the fluorinated misonidazole analogue.

19F-magnetic resonance spectroscopy (MRS) can be used to non-invasively monitor metabolism of 5-fluorouracil (5FU) to cytotoxic fluoronucleotides (FNuct). We investigated whether the levels of FNuct formed from 5FU and observed in vivo by MRS in the Walker carcinosarcoma predicted cytotoxicity. Fifty mg kg-1 5FU caused tumour FNuct formation and, when repeated daily for 1 week, significant tumour growth inhibition (P less than 5%). Twenty-five mg kg-1 5FU produced less tumour FNuct (P less than 5%) and did not cause significant tumour regression. Tumour regression and tumour FNuct formation were also suppressed by 50 mg kg-1 5FU combined with a molar equivalent dose of allopurinol (P less than 2%). Tumour extracts were analysed by hplc and MRS confirming the observations in vivo and demonstrating that peak integrals in vivo were directly proportional to 5FU and FNuct concentrations. Hplc analysis of extracts showed that 50% of FNuct in tumours treated with 5FU was the cytotoxic nucleotide FUTP; this was lowered to 5% by a molar equivalent dose of allopurinol (P less than 2%). Twenty-five mg kg-1 5FU also produced significantly less FUTP (36%) than the 50 mg kg-1 dose (P less than 5%). These results suggest that MRS-detectable changes in tumour FNuct (mostly in FUTP) can be used to predict 5FU cytotoxicity.

A study of the healing of peritoneal defects in the rat in the presence of the Walker 256 tumour has been made. The healing process was investigated histologically, by autoradiography, and by hydroxyproline estimation of the healing peritoneal wound. There was no difference in the rates or quality of the healing process in the control or tumour bearing animals.

The effect of the Walker 256 rat carcinoma, growing at a site remote from the liver, on liver glycogen synthesis has been determined. Although the mechanism has not been defined it has been shown that the deposition of liver glycogen in response to a glucose load is inhibited in tumour bearing rats. It has been further demonstrated that, by comparison with the normal liver, the liver depleted of glycogen is more susceptible to the development of metases from circulating cancer cells.

Normal and tumour tissues from rats, blood from normal and tumour bearing rats, and normal human blood were examined using the electron paramagnetic resonance (epr) technique. At low temperature a triplet epr signal, which is known to be produced by a NO-haemoprotein complex, was detected in some tumour samples and in decaying normal liver. At room temperature all of the tumour samples examined gave a doublet signal. This signal was also detected in blood but not in other normal tissues. The signal has a g value of 2·0054 ± 0·0002 and a hyperfine splitting of 1·80 ± 0·05 G and is assigned to the ascorbyl free radical. Model experiments suggest that the appearance of detectable concentrations of this radical result from a disturbance of the normal state of the ascorbic acid, dehydroascorbic acid redox system. It was verified that cell division is not responsible for the ascorbyl radical although autolysis may be involved. A possible relationship between the formation of ascorbyl radicals and other paramagnetic species in tumours is discussed.

Exocrine cells of the pancreas of male rats bearing the Walker carcinoma show a striking accumulation of stainable neutral lipid in the form of small aggregated droplets in the base of the cells. In several cases, epithelial cells of small ducts also contained fat. Stainable lipid is sometimes present in cells of the pancreas of normal rats and in rats in which the Walker tumour has failed to grow: lipid in duct cells was confined to tumour-bearing animals.