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The antitumorigenic effects of carotenoids, in addition to their immuno-enhancing effects, may occur by their direct action on growing tumor cells. To test this hypothesis the direct inhibitory effect of various concentrations of canthaxanthin (CX; 4,4'-diketo-beta-carotene), a non-provitamin A carotenoid, was tested on the in vitro growth of JB/MS, B16F10 melanomas and PYB6 fibrosarcoma and murine non-transformed NIH-3T3 (ATCC CRL 1658) cells. At concentrations of 1 x 10(-8) M up to 1 x 10(-4) M, CX significantly reduced the overall number of tumor cells. The greatest inhibition was observed at a CX concentration of 1 x 10(-4) M after 72 h and 96 h of incubation. However, CX had no inhibitory effect on the growth of the non-transformed NIH-3T3 cell line; rather it significantly enhanced growth of this cell line (P less than 0.05) after 96 h of incubation. Thus, the inhibitory action of CX on growing tumor cells appears to be due to its direct actions on tumor cells and not via its conversion to vitamin A or its immuno-enhancing effects.

GR9 is a chemically induced fibrosarcoma composed of clones with different H-2 class-I expression. These clones differ with respect to local growth and spontaneous metastasis. The B9 clone (H-2 negative) is highly tumorigenic (local growth) but of low metastatic potential (spontaneous metastasis assay). We have analyzed the effect that transfection of H-2Dd and H-2Kd genes on this clone have upon local growth, lung colonization after i.v. injection and ability to form spontaneous metastases. The results showed that the effect on local growth of transfection of the Kd-gene was stronger than that of the Dd gene. In addition, B9 co-transfected with H-2Kd and Dd genes showed the highest immunogenic properties in syngeneic BALB/c mice. Interestingly, the pSV2-neo transfected clone gave almost the same result as that obtained with Dd transfection. Lung colonization after i.v. injection of the different clones (experimental metastasis), paralleled the results obtained for local growth: the number of lung nodules followed the cadence KdDd less than Kd less than Dd less than pSV2. Survival of mice was always inversely correlated with local growth, e.g., all mice injected with 5 x 10(5) B9 H-2KdDd transfected cells survived. In contrast, no mice injected with the B9 control did. These differences were abrogated in irradiated and nude BALB/c mice. Finally, all transfected clones remained non-metastatic in a spontaneous metastasis assay, behaving as the control, non-transfected B9 cells.

We have previously reported that the lethal toxicity of 5-fluorouracil (5-FU) in specific-pathogen-free mice is due to an indigenous infection with Escherichia coli (K. Nomoto, T. Yokokura, Y. Yoshikai, et al. Can. J. Microbiol. 37:244-247, 1991). In the present study, we demonstrate that nonspecific immunostimulation augments host resistance against the lethal toxicity of 5-FU in tumor-bearing mice. Intravenous administration of a preparation of heat-killed Lactobacillus casei (LC 9018), a nonspecific immunostimulant, at a dose of 20 mg/kg to BALB/c mice augmented their resistance against the lethal toxicity of 5-FU if the preparation was injected into the mice 10-40 days before administration of 5-FU. Injection of LC 9018 into BALB/c mice bearing Meth A fibrosarcoma also enhanced their resistance against the lethality of 5-FU. Systemic infection with E. coli was induced in all of the 5-FU-treated tumor-bearing mice 10 days or more after administration of the drug at a lethal dose of 500 mg/kg, and it was accompanied by an overgrowth of the bacteria in the intestine. Treatment of tumor-bearing mice with LC 9018 resulted in decreased rates of occurrence of systemic infection with E. coli and inhibition of overgrowth of the bacteria in the intestine after administration of 5-FU. A single administration of either LC 9018 or 5-FU significantly inhibited the growth of Meth A cells in vivo, and a combined antitumor effect was shown in the mice treated with both 5-FU and LC 9018.

The antitumor effect of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received intradermal inoculations of syngeneic Meth-A fibrosarcoma in the right (primary tumor, 10(6) cells) and left (distant tumor, 2 x 10(5) cells) flanks. Intratumoral administration of PSK significantly inhibited the growth of not only the right but also the left tumor. PSK also inhibited the growth of a methylcholanthrene-induced fibrosarcoma BAMC-1, and a methylurethane-induced adenocarcinoma Colon 26 in the double grafted tumor system of syngeneic BALB/c mice. However, when the left distant tumor was different from the right Meth-A tumor, the intratumoral administration of PSK in the right tumor was unable to inhibit the growth of the left BAMC-1 or RL male-1 tumor. The PSK-induced immunity, therefore, is tumor-specific and T lymphocytes may play an important role in antitumor memory function. The enhancement of concomitant immunity by PSK treatment was completely impaired by previous intravenous administration of an alkylating agent, cyclophosphamide (CY). The enhancement of sinecomitant immunity by PSK treatment was also impaired by previous CY intravenous administration. The antitumor effect of PSK was suppressed by previous intravenous administration of another alkylating agent, ACNU. It is possible that alkylating agents suppress the function of effector T cells and granulocytes which are very important for the antitumor immune cascade reaction due to PSK treatment. On the other hand, the antitumor effect of PSK was enhanced by previous intravenous administration of an anti-metabolite, 5-fluorouracil.

We studied the therapeutic effects of adoptive immunotherapy with lymphokine-activated killer (LAK) cells combined with chemotherapy on BMT-11 fibrosarcoma in C57BL/6 mice. Compared with the untreated group, no significant therapeutic effect was brought about by CY therapy alone or LAK.rIL-2 alone and all mice belonging to these three groups died with a mean survival time (MST) of 45.3, 51.8 and 45.9 days respectively. CY plus LAK.rIL-2 brought about complete cures in 3 out of 8 mice (37.5%) and a significant prolongation of MST of mice which died (64.4 days) and the accumulation of LAK cells (% Dose/g) at tumor sites was enhanced more than 7-fold by combination with CY. On the other hand, the therapeutic effects of cytotoxic T lymphocytes (CTLs) was sufficiently high even in the CTL.rIL-2 alone and were only slightly enhanced by combination with CY compared with LAK cells. Also, we detected LAK-attractant activity in the conditioned medium (CM) of CY-treated tumor tissues but not in that of untreated tumor tissues, and peak activity was reached 5 days after CY-treatment. This attractant activity was located in two major 10,000-50,000 M. W. fractions of CM. We then observed that LAK-attractant was produced in CM of host reactive cell enriched fractions from CY-treated tumor tissues, but not in that of tumor cell enriched fractions. The above findings imply that the effects of adoptive immunotherapy depend upon the accumulation of transferred effector cells at tumor sites, and we believe that the production of LAK-attractant by tumor tissue, facilitated by chemotherapy, is one of the mechanisms responsible for enhanced LAK-cell-accumulation at tumor sites. We performed a preliminary clinical trial with adriamycin, autologous spleen-LAK cells and rIL-2 on 30 hepatocellular carcinoma patients after radical resection on the basis of the experimental results above. There were no significant therapeutic effects after adoptive immunotherapy during the postoperative course but tendency for temporary inhibition of recurrence. Thus it is shown that this method has probable value as effective adjuvant postoperative therapy.

A number of directly acting carcinogenic N-nitroso compounds were administered to female F344 rats intravesically, to assess their ability to induce tumors locally in the urinary bladder and systemically following absorption through the bladder mucosa. The compounds were alkylnitosoureas and alkylnitrosocarbamates and could be formed by interaction of amides with bacterially produced nitrite in infected bladders. Methylnitrosourethane was very toxic: doses of 1-2 mg caused death of some rats. A total dose of 0.15 mmol of ethylnitrosourethane, which was much less toxic, was administered to each rat and almost all developed bladder tumors. Ethylnitrosourea also gave rise to bladder tumors following intravesical treatment, and induced some tumors systemically, whereas methylnitrosourea, 2-methoxyethylnitrosourea and 2-hydroxypropylnitrosourea induced bladder tumors in high incidence and few tumors systemically. Nitrosooxazolidone was quite toxic and induced few bladder tumors. The dialkylnitrosoureas were more stable and some induced more tumors systemically than the monoalkylnitrosoureas. 1,3-Dimethylnitrosourea induced no bladder tumors and 1,3-diethylnitrosourea very few, but both induced tumors systemically that were similar to those induced by gavage treatment of rats. 1-Ethyl-1-nitroso-3-hydroxyethylurea, 1-hydroxyethyl-1-nitroso-3-ethylurea and 1-(2-hydroxypropyl)-1-nitroso-3-(2-chloroethyl)-urea induced bladder tumors in a majority of rats treated intravesically; the first induced many tumors systemically. Most of the bladder tumors were transitional cell papillomas and carcinomas, but there were a few squamous cell tumors, smooth muscle tumors, sarcomas and carcinosarcomas. The effects of intravesical administration of the directly acting alkylating compounds are compared with the effects of similar doses given to rats by gavage.

We have previously shown that the combination of immunotherapy with interleukin 2 (IL-2) and local hyperthermia (LHT) abrogates the growth of murine subcutaneous tumors significantly more than either modality alone. This study was undertaken to investigate whether the beneficial effect of combined modality therapy could be attributed to increased trafficking of effector cells to the tumor. After inducing MCA-105 sarcomas in the hindlimbs of C57BL/6 mice, animals were given no therapy, LHT, IL-2, or IL-2 + LHT followed by an iv injection of 51Cr-labeled syngeneic splenocytes or LAK cells. Select organs and the tumor-bearing extremity were counted in a gamma counter. IL-2 or LHT alone did not affect lymphocyte migration, while IL-2 + LHT significantly decreased trafficking (P less than 0.001) to the tumor. LAK cells showed increased migration to the tumor site compared to splenocytes in all treatment groups (P less than 0.02). IL-2 caused increased migration of LAK cells but not splenocytes to the lung; this was not affected by LHT. LAK cell trafficking to the spleen was decreased by IL-2 and IL-2 + LHT, while splenocyte migration was decreased in the LHT and combined treatment groups. LHT and IL-2 had no effect on trafficking of either effector cell type to liver or kidney. These results show that the beneficial effect of combined modality therapy may not be due to increased trafficking of lymphocytes to the tumor area. In addition, LAK cells traffic more to subcutaneous tumors than splenocytes, and this cannot be explained by the differential trafficking to other organs. The results of this study will be important in the planning of future experiments with combined adoptive immunotherapy and hyperthermia.

A fluorine-18 labeled analogue of D-talose, 2-deoxy-2-[18F]fluoro-D-talose ([18F]FDT), was synthesized via nucleophilic fluorination with [18F]fluoride ion and its biodistributions in animals were examined. Radiofluorination of benzyl 3,5,6-tri-O-benzyl-2-O-(trifluoromethanesulfonyl)-alpha-D-galac tof uranoside (5) with aminopolyether supported potassium [18F]fluoride (K18F/Kry222) in acetonitrile followed by deprotection of the [18F]fluorinated intermediate (6) with boron tribromide in CH2Cl2 gave [18F]FDT in an average radiochemical yield of 29% with a radiochemical purity greater than 98%. Biodistribution studies of [18F]FDT in mice bearing fibrosarcoma showed the highest uptake of radioactivity in the liver (34.9% dose/g), followed by the kidney (15.9%dose/g), the small intestine (12.9%dose/g) and fibrosarcoma (5.7%dose/g), at 30 min after i.v. administration. Although the radioactivity in the kidney and small intestine decreased with time, the uptake in the liver and the tumor slightly increased until 120 min. The high liver uptake of [18F]FDT was also observed in normal rats and this uptake was strongly inhibited by co-administration of D-galactose. These preliminary results suggest that [18F]FDT might be metabolized through the galactose metabolic pathway as analogously observed with 2-deoxy-2-[18F]fluoro-D-galactose which is an isomer with respect to carbon-2 of [18F]FDT, and that it may be another candidate for studying liver function by positron emission tomography.

BALB/c mice subjected to water stress twice daily, between 3 and 7 min at each exposure 3 days before, 5 days after, or 3 days before and 5 days after intradermal Meth A tumour transplantation, showed an increase of tumour size and tumour growth rate compared with that of home cage control (HCC) mice. However, BALB/c mice subjected to water stress 3 days before and 6 days after tumour transplantation showed a decrease of tumour size and tumour growth rate compared with HCC mice. BALB/c mice subjected to 2 min of handling stress daily on days 1 through 5, but not BALB/c mice subjected to handling stress on days 1 through 7 or on days 6 through 7 of tumour growth, showed an increased in tumour size relative to HCC mice. On the other hand, BALB/cBy mice handling stressed on days 1 through 7, but not BALB/cBy mice handling stressed on days 1 through 5 or days 6 through 7 after tumour transplantation, showed a decreased tumour size compared with HCC mice. No effect of stress could be observed when nu/nu BALB/c mice were subjected to water stress 3 days before and 6 days after tumour transplantation. However, BALB/c nu/nu mice pre-injected intraperitoneally with spleen cells from conventional mice had a decreased tumour size and growth rate of the tumour when stressed 3 days before and 6 days after tumour transplantation.

Metastatic nodules were examined by DNA fingerprint analysis. The probes used, Pc-1 and Pc-2, detect mutations as shifts in bands of the minisatellite loci which are dispersed among chromosomes. Four clonal lines of a fibrosarcoma from an F1 mouse (C57BL/Ka x C3H/He) were selected for various metastatic potentials upon inoculation into syngeneic mice. These four lines exhibited many extra bands resulting from recombination and/or DNA slippage, indicating accumulation of mutations during the successive passages in mice. One of the four, a 505 cell line which had been passaged extensively in vitro and consisted of a heterogenous population, was inoculated into thirteen syngeneic mice, and gave rise to six lung metastatic nodules in two mice. All the nodules showed band-patterns distinct from one another, although nodules within a given mouse tended to show similar patterns. When a genetically tagged 505-05-01 clone was analyzed, three of nine metastatic nodules obtained also revealed new bands. These results strongly suggest that somatic mutations occur at a high frequency during metastasis, providing direct evidence of genetic instability of the tumor cells.

The intracellular degradation of newly synthesized collagen was studied in both normal fibroblast and 3-methylcholanthrene induced fibrosarcoma cells. The degradation of newly synthesized collagen was examined using pulse-chase experiments and radioactive labelling techniques with [3H]-proline. The percentage of intracellular proteolysis of newly synthesized collagen was determined by measuring the formation of [3H]-hydroxyproline containing fragments in alcohol-soluble and insoluble fractions of normal cells and fibrosarcoma cells in the culture. The rate of degradation of newly formed collagen was then followed by estimating the radioactivity of [3H]-hydroxyproline at different intervals, during the chase period. The results clearly demonstrated that the percent of intracellular degradation of newly synthesized collagen was approximately three fold higher in fibrosarcoma cells than in normal fibroblast cells. The increased intracellular degradation of newly formed collagen was followed by an increase in the activity of cathepsin B and L in fibrosarcoma cells. The pulse-chase experiments indicated that the rate of degradation of newly synthesized collagen in fibrosarcoma cells is relatively greater than in normal fibroblast cells. In addition, as the labelling time increased, the formation of [3H]-hydroxyproline containing peptides in the ethanol-soluble fraction were found to be increased in both normal cells and fibrosarcoma cells, but the extent of formation was higher in fibrosarcoma cells compared to normal fibroblast cells. The results of this investigation collectively suggest that the intracellular degradation of newly synthesized collagen is enhanced in fibrosarcoma cells.

The in vivo antitumour activity of a beta 1----3/1----6 glucan from the fungus Glomerella cingulata was investigated in vivo. The glucan exhibited a strong inhibition of tumour growth of the allogeneic Sarcoma-180 as well as the syngeneic DBA/2-MC.SC-1 fibrosarcoma with inhibition ratios up to 100%. Against the hormone sensitive Noble-Nb-R prostate carcinoma the glucan alone showed a moderate antitumour effect, whereas in combination with diethylstilbestrol an almost complete regression of the tumour could be achieved. It could be demonstrated that a highly ordered structure of the glucan is not essential for the antitumour activity. Since the glucan expressed no direct cytotoxic effects, the immunomodulating activity was investigated in vitro in order to get an indication for a possible mode of action. In the lymphocyte transformation assay the glucan at a dose of 100 micrograms/ml caused a fourfold increase in the proliferation of murine spleen lymphocytes. Moreover, the glucan stimulated the phagocytosis of zymosan by bone marrow macrophages up to 100%. However, the glucan was not able to render macrophages cytotoxic against P-815 mastocytoma cells.

MY-1, which consists of DNA and RNA extracted and purified from bacillus Calmette-Guérin (BCG), has been shown to have strong antitumor activity against various experimental tumors. To examine the role of T cells in the antitumor mechanism of MY-1, the effect of MY-1 injection on the development of tumor-specific immunity against MethA fibrosarcoma was investigated. MY-1 injections inhibited tumor growth less effectively in T-cell-deficient nude mice than in normal BALB/c mice. MethA tumor growth was suppressed after inoculation with L3T4-positive lymphocytes from tumor-bearing mice treated with MY-1. MethA-specific delayed-type hypersensitivity was also detected in tumor-bearing mice treated with MY-1. Immunohistochemical analyses showed that many L3T4-positive and a few Lyt2-positive cells infiltrated the regressing tumors. These results indicate that intratumoral MY-1 injections induce a MethA-specific, L3T4-positive cell-mediated, delayed-type hypersensitivity, which is necessary for the tumor regression.

DNA topoisomerase I, a nuclear enzyme important for solving topologic problems arising during DNA replication, has been identified as a principal target of a plant alkaloid, 20(s)-camptothecin. In view of the profound biochemical effects of camptothecin and its analogues on DNA replication and the differential cytotoxic effects on human tumors in xenografts, experiments were performed to determine whether topotecan, a camptothecin analogue, would potentiate the radiation effects on human carcinoma cells in culture and murine fibrosarcoma in mice. Cell culture studies showed that a dose dependent reduction in cell survival was obtained with a 4 hr exposure of the drug following irradiation of cells. No enhancement of cell killing was seen when cells were treated with the drug before irradiation. Preliminary in vivo tumor studies showed a significant radiosensitizing effect of topotecan that was both drug dose (20 mg/kg) and time sequence (4 hr before irradiation) dependent. There was no enhanced skin reaction following the combined treatments.

Brain catecholamines (CA) were studied in discrete brain areas of benzo(a)pyrene (b(a)p) induced fibrosarcoma bearing mice. Dopamine (DA) and norepinephrine (NE) levels decreased significantly in different brain areas especially in corpus striatum and hypothalamus with the tumor progression, indicating an inverse relationship between brain DA and NE levels and tumor growth. Since impaired hormonal and immunological functions are manifestation of systemic alteration during tumor growth, it appears that during malignant growth an alteration of these brain CA may play an important role in the regulation of systemic alterations.

The biological stains, methylene blue and its metabolite azure B, were evaluated as anti-tumor and anti-inflammatory agents. Azur B, administered in drinking water to tumor-bearing mice, inhibited the growth of transplanted tumors and the growth of primary tumors induced by methylcholanthrene. Inhibition of growth of primary tumors was observed only in female mice. Azure B also reduced the wet weight of carrageenin-induced granulomas in rats. Azure B, given intravenously to BCG-sensitized mice 15 minutes prior to challenge with lipopolysaccharide, decreased TNF production (to 10% of control values) and prevented death from endotoxic shock. Methylene blue decreased TNF production (to 50% of control values) but did not protect the animals from endotoxic shock. Our results suggest that some of the effects previously ascribed to methylene blue are probably mediated via its metabolite, i.e. azure B. Low toxicity and easy administration of the dyes explain their use in clinical settings.

Multinuclear nuclear magnetic resonance spectroscopy was used to follow the metabolism and kinetics of 5-fluorouracil (5FU) after i.v. administration at a dose of 100 mg/kg on Wistar rats. 31P spectra allow one to determine both the energetic status and the pH of the tissues under investigation, while serial 19F spectra reveal the drug clearance. Analyses of 5FU kinetics show that the half-life of 5FU elimination is about 35 min in tissue with a pH of 7.3. However, this half-life increases 2.5-fold when the local pH decreases below 6.9. Thus, acidification seems to induce a local retention of 5FU, which tends to prove the existence of active transport. This retention of the drug may have significant clinical implications for assessing and improving chemotherapy alone or in combination.

We have used the murine 3-methylcholanthrene induced T10 fibrosarcoma tumor cell system originating in (C3II/en x C57BL/6)F1 mice (H-2b x H-2k) to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non metastatic and NK sensitive IC9 cells (Db+, Kk, Kb, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumor cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Kk-, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Transfection of Dk negative variants with the H-2Dk gene, resulted in the isolation of several clones which expressed a wide range of metastatic phenotypes but maintained sensitivity to NK. In addition, by cloning the cDNA of the H-2Dk gene of the metastatic T10-IE7 variant cells and analyzing its nucleotide sequence, we found four single nucleotide changes. Two of them are not expected to alter the encoded amino acids, whereas the others should result in two amino acid substitutions in the alpha-2 domain of the class I H-2Kd protein product. These changes might account, at least partially, for the failure of the transfection of H-2Dk to restore resistance to NK.(ABSTRACT TRUNCATED AT 250 WORDS)

Echitamine chloride a plant alkaloid from Alstonia scholaris has been used to examine the anticancer effects on methylcholanthrene-induced fibrosarcoma. Echitamine chloride dissolved in saline (10 mg/kg body weight) and injected subcutaneously for 20 days in fibrosarcoma rats has exhibited significant regression in tumor growth. The altered activities of plasma and liver transaminases and gamma-glutamyl transpeptidase and lipid peroxidation in fibrosarcoma have been corrected to near normal after echitamine chloride treatment. The decreased liver glutathione content and the lowered activities of glutathione peroxidase, superoxide dismutase and catalase have also been reversed to near normals after echitamine chloride treatment.

When soybean oil containing tocopherol acetate was given to rats once a week subcutaneously for 10-12 months, it caused the development of fibrosarcomas at the injection site in 11 of 15 rats. A tumor produced in this manner proved eminently transplantable into other rats. The molecular species of phospholipid subclasses were determined in primary and transplanted tumors. The molecular species composition of the phospholipid subclasses in both types of tumors were similar. The percentages of diacyl and alkylacyl glycerophosphocholine (GPC) were 90-93 and 6-8% of total phosphatidylcholine, respectively. The percentages of diacyl and alkenylacyl glycerophosphoethanolamine (GPE) were 51 and 45%, respectively, of total phosphatidylethanolamine (PE). Diacyl and alkylacyl GPC species containing arachidonic acid (20:4) composed about 15-16 and 37-40% of each subclass, respectively. Diacyl and alkenylacyl GPE species containing 20:4 composed about 38-40 and 56-60% of each subclass, respectively. Disaturated species of diacyl and alkylacyl GPC composed about 22-24 and 13% of each subclass, respectively, whereas these species of PE composed less than 2%. The fatty acid composition of the other tumor phospholipids was analyzed.

Patterns of p53 expression were investigated in chemically induced fibrosarcoma tumors and cell lines. Most, if not all, cell lines were found to carry alterations at the protein level, reflected in the overproduction of greatly stabilized p53 proteins. In many cases, this was accompanied by formation of complexes with hsc70. Hence, all of these lines may be expressing one sort or another of mutant p53. The mutant nature of the p53 gene was directly verified, in a number of cases, by PCR-amplified cDNA cloning. In one line, no p53 protein was made at all; this turned out to be because of a mutation in a splice donor site, resulting in the production of an aberrant mRNA. In all other cases, mRNAs carrying mis-sense mutations were present, and were sometimes expressed along with wt p53 mRNA. When tested in an in vitro transformation assay, all cloned mutants possessed a discrete oncogenic activity, while having lost the ability to interfere with oncogene-mediated transformation. The system described here could potentially be very helpful in elucidating the significance of p53 mutations.

Cyclosporine A (CsA) was used to immunosuppress male Sprague-Dawley rats treated with the chemical carcinogen 3-methylcholanthrene (3MC). Rats treated with low doses of CsA (2.5 or 5 mg/kg) given 2 days prior to an injection of 3MC, and then daily for 2 weeks or twice weekly for 10 weeks did not develop tumors. Rats treated with 2.5 mg/kg CsA for 2 weeks beginning 5 days after a single 3MC injection had tumor incidence similar to rats treated with 3MC only. To further examine the effects of CsA on immune function, groups of rats were then treated with 2.5, 5, 10 or 20 mg/kg CsA daily for 14 days and immune function assessed by measuring delayed-type hypersensitivity (DTH), natural killer cell (NK) activity, and production of interleukin 2 (IL-2), interferon (IFN), prostaglandin E2 (PGE2) and specific IgG antibody. Natural killer cell cytotoxicity was enhanced and antibody production was suppressed in rats treated with all doses of CsA tested. Interleukin 2 production was elevated at the two lower doses, but antibody production, DTH reactions and synthesis of IL-2 and IFN were suppressed with the higher dose treatments (10, 20 mg/kg CsA). The enhanced NK activity seen in rats treated with the lower doses of CsA may be due to the increase in IL-2 production, while enhancement of NK activity at higher doses may be due to other mechanisms. The tumor data suggest that CsA does not prevent tumor formation in our chemical-induced model due to an increase in NK activity, since this enhancement was seen even when tumors did develop normally.

The carcinogenic effects of cadmium have not been thoroughly assessed in the commonly used Fischer (F344) rat. This study determined tumor incidence in various tissues of male F344/NCr rats after a single dose of cadmium. Cadmium (as CdCl2) was given sc in the dorsal thoracic midline at 30 mumole/kg to 70 8-week old male F344 rats while controls (n = 50) received saline. Rats were observed during the next 90 weeks. Early deaths (less than or equal to 32 weeks), due mostly to acute cadmium-induced hepatotoxicity, accounted for 37 of the cadmium-treated rats while no control rats died in the same period. A high incidence of injection site sarcomas (ISS) occurred in the cadmium-treated group (21 ISS/32 rats at risk; 66%) while only 1/50 occurred in controls (2%). In fact, ISS were the major cause of morbidity after 35 weeks in cadmium-treated rats. These tumors were mostly fibrosarcomas, although histiocytic and osteogenic sarcomas also occurred. Testicular interstitial cell tumors, which show a very high spontaneous incidence in this strain (41/49; 84%), were not markedly affected by cadmium (30/31; 97%). This is in sharp contrast to other strains, such as the Wistar, in which cadmium treatment is reported to cause as much as an eightfold increase in interstitial cell (Leydig cell) tumor incidence. The incidence of large granular lymphocyte (LGL) leukemia, which also occurred frequently in control F344 rats (12/47; 26%) was markedly decreased (2/31; 7%) by cadmium. Our recent studies indicate acute lymphonecrotic effects occur with cadmium in lymphoid tissues of rats, and this may be related to the suppression of the leukemia. These results indicate that cadmium is very effective in inducing ISS in F344 rats, as is the case with other strains thus far tested, and also markedly reduces spontaneous leukemia incidence.

Recent studies have shown that ultraviolet (UV) irradiation induces a systemic effect which enhances subsequent tumor induction by benzo[a]pyrene in a manner which is dependent on the dose of benzo[a]pyrene. The present study was designed to test whether UV-B irradiation renders mice susceptible to subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene. The sources of UV-B irradiation were banks of 6 Westinghouse FS-40 sunlamps, situated 20 cm above the mouse cages. Female BALB/cAnNHsd received five 30-min dorsal UV-B radiation treatments per week for 12 weeks, resulting in a total dose of approx. 6.4 x 10(5) J m-2. Two to seven days after termination of UV treatments, syngeneic regressor tumor cells (BP2) induced by benzo[a]pyrene were injected subcutaneously or intravenously into irradiated mice and unirradiated controls. By 38 days post subcutaneous implantation, 24/30 and 3/30 BP2 implants were detectable in the irradiated and unirradiated mice, respectively. Ultraviolet irradiated mice were also unable to reject lung colonies resulting from intravenous administration of BP2 cells, although they were rejected by unirradiated mice. The mean number of lung colonies per mouse was 16- to 35-fold greater in UV irradiated mice than in unirradiated controls, at 14 to 17 days post injection. Thus, UV irradiation rendered mice, with no known exposure to benzo[a]pyrene, susceptible to a subcutaneous or intravenous injection of a regressor tumor induced by benzo[a]pyrene.

Natural vitamin E and synthetic vitamin E (dl-alpha-tocopheryl acetate) were tested for their tumorigenicity in rodents. Transplantable tumors, at the site of injection, were induced by repeated injections of these compounds in two strains of mice, NFS/N and C57BL/6N x C3H/He F1, and in a strain of rats, Fischer 344. Natural vitamin E was tumorigenic in both strains of female mice only when injected with soya oil. In contrast, dl-alpha-tocopheryl acetate alone was capable of inducing tumors in Fischer 344 rats. Only one out of 5 male NFS/N mice given dl-alpha-tocopheryl acetate developed a tumor. Therefore, Fischer 344 rats were more susceptible to tumor formation by dl-alpha-tocopheryl acetate than NFS/N mice. dl-alpha-Tocopheryl acetate with soya oil or with palm oil also resulted in the formation of transplantable tumors in NFS/N mice and Fischer 344 rats. There was no difference in the tumor incidence between mice treated with dl-alpha-tocopheryl acetate alone and dl-alpha-tocopheryl acetate plus soya oil or palm oil. However, in rats, the incidence was lower for a group treated with dl-alpha-tocopheryl acetate plus palm oil than for those with dl-alpha-tocopheryl acetate alone and with dl-alpha-tocopheryl acetate plus soya oil.

A monoclonal IgM antibody, H17, has been obtained from rats immunized with mouse fibrosarcoma cells from an in vitro established methylcholanthrene (MCA)-induced tumour. H17 shows specific and very selective binding to alpha-N-acetylgalactosamine (GalNAc alpha) when tested for reactivity to a panel of glycolipids. It cross-reacts with GalNAc alpha on the Forssman antigen extracted from dog small intestine, but not from the human blood group A determinant, a finding not commonly observed among antibodies with this specificity. Despite its specificity, H17 does not react with TA3-Ha, a mouse mammary adenocarcinoma, known to express the Tn antigen (GalNAc alpha-O-Ser/Thr). The uniqueness of H17 probably relates to the fact that it has been generated against an MCA-induced tumour rather than against the pure saccharide itself. Minimum energy conformation structures of different GalNAc alpha containing saccharide molecules were computer modelled to allow a plausible interpretation of the accessible site of GalNAc alpha for successful interaction with the H17 paratope as compared to other GalNAc alpha binding antibodies.

Tumor-draining lymph node cells from mice bearing the methylcholanthrene-induced MCA 106 tumors can be sensitized in vitro to acquire antitumor reactivity. We examined the effect of interferon alfa on the function of cells that underwent in vitro sensitization in adoptive immunotherapy. Interferon alfa increased the antitumor reactivity of in vitro sensitized cells in the treatment of MCA 106 pulmonary metastases. This effect was evident in irradiated mice, indicating that a host response to the interferon alfa was not required. Interferon alfa treatment increased class I major histocompatibility complex antigen expression on tumor cells and increased their susceptibility to lysis by in vitro sensitized cells. These results suggest that interferon alfa enhancement of adoptive immunotherapy was mediated by its effect on tumor cells. Interferon alfa may be a useful adjunct to the adoptive immunotherapy of human cancer.

Fast-growing tumors are major glutamine consumers and may alter host glutamine metabolism to benefit the tumor. Previous studies from our laboratory have demonstrated that the liver switches from an organ of glutamine balance to one of glutamine release with progressive malignant growth. However, the regulation of this change is unclear. This study examined tumor modulation of hepatic glutamine metabolism by determining the activities of glutaminase, the principle enzyme of glutamine degradation, and glutamine synthetase, the principal enzyme of glutamine synthesis. Hepatic glutamine content was also determined. Rats with a fast-growing subcutaneous fibrosarcoma (TBR) and pair-fed controls were studied at 2 and 3 weeks after tumor or sham implantation, when the tumors comprised approximately 5% and 20% of total body weight. Arterial glutamine fell with progressive tumor growth (608 +/- 26 mumol/L in controls vs 494 +/- 15 in TBR, p less than 0.005) and was not attributable to a diminished food intake. Hepatic glutamine content was increased 45% (p less than 0.01) in tumor rats at 2 weeks due in part to a 35% fall in liver glutaminase activity. At 3 weeks, glutamine synthetase activity increased by 43% (0.58 +/- 0.07 mumol/mg of protein/hr in controls vs 0.83 +/- 0.04 in TBR, p less than 0.01) whereas glutaminase remained depressed (2.68 +/- 0.12 mumol/mg of protein/hr in controls vs 2.22 +/- 0.15 in TBR, p less than 0.05) and glutamine content fell compared to 2 week tumor-bearing rats, consistent with accelerated hepatic glutamine release. Tumors may alter liver glutamine metabolism by modulating hepatic enzyme activity in order to provide circulating glutamine for the growing malignancy.

The DNA of 22 fibrosarcomas, newly induced in BALB/c mice by subcutaneous doses of 3-methylcholanthrene (3-MCA), was tested in NIH 3T3 transformation assay. Activation of K-ras and N-ras was found in 7 and 3 cases respectively. No H-ras activation was detected. Polymerase chain reaction and oligonucleotide hybridization performed on the DNA of the 22 sarcomas revealed 5 cases of K-ras mutation at codon 12, 3 at codon 13 and 1 at both codons. One case of K13 mutation was not detectable by transfection. Three cases of mutation at codon 61 of N-ras were also found, one of which was simultaneous with a K12 mutation. Tumor-specific transplantation antigens (TSTA) were assessed in the 22 original tumors. Altogether 16 sarcomas were immunogenic, with the highest frequency of TSTA+ tumors (10/11 and 5/6) in the groups given 1.0 and 0.1 mg of 3-MCA respectively, the lowest (1/5) in that with 0.01 mg of carcinogen; ras mutations occurred in the DNAs of 11 out of the 16 TSTA+ sarcomas, but none of the DNAs of the 6 TSTA- tumors showed ras mutation. The results suggest that 3-MCA-induced transformation of subcutaneous fibroblasts can involve mutations in codons 12, 13 or 61 of K- and N- but not H-ras gene and that such mutation is accompanied by the expression of TSTA.

It has been found that the methylcholanthrene-induced rat fibrosarcoma contains an enzyme (probably a cathepsin) which digests type I and type III collagens in acid pH. At physiological pH no proteolytic activity against collagen was found. It may be concluded that the tumour collagen is degraded mainly by the action of cathepsin(s).

The in vitro bulk electrical properties of MCA1 fibrosarcoma induced in C57B1/6 male mice were measured at frequencies of 10 kHz to 100 MHz, with some tissues measured to 2 GHz. The properties of normal surrounding tissue also were measured. A comparison of the dielectric properties between three different stages of tumor development as well as that between various locations within the tumor is reported. Statistical analysis of the experimental results revealed statistically significant differences in the dielectric constant and conductivity of the tumor tissues at various stages of development as measured at frequencies below 10 MHz. Conductivity values at different stages also differ at a frequency of 100 MHz. At other frequencies these differences were found to be statistically insignificant.

Intraperitoneal (i.p.) injection of 7% Sheep Erythrocytes (SRBC) was found to inhibit growth of a transplanted tumour and exhibit a consequent increase in total survival compared to untreated tumour controls. In an attempt to probe into the mechanism(s) involved, functional aspects of Polymorphonuclear Neutrophils (PMNs) were investigated in terms of their 'metabolic (respiratory) burst' during tumour phagocytosis. Both NADPH-Oxidase mediated superoxide anion (O2-.) formation (NBT reduction) and Myeloperoxidase (MPO) mediated oxidisable halide incorporation (131I incorporation) were found to be highly stimulated by SRBC in normal (CS) and tumour bearing counterparts (TS). A little tumour mediated residual inhibition persists on the MPO system in PMNs of animals with tumour plus SRBC (TS) which, however, showed an obvious bonus effect over the tumour controls (TC). The results suggest a possible mechanism of tumour inhibition by SRBC in mice with the involvement of highly stimulated phagocytic metabolism in PMNs.

We analyzed the H-2 class-I expression of different clones obtained from 4 different methylcholanthrene-induced tumors in BALB/c (H-2d) mice. The results clearly indicated high intra-tumor heterogeneity in all 4 fibrosarcomas with regard to H-2 K, D and L expression. Clones were found to be H-2-negative, H-2-positive, or to present intermediate expression. Southern blot analysis of class-I genes showed RFLPs equal to those obtained from normal BALB/c DNA, ruling out rearrangements or gross deletions in the class-I genes of different tumors. However, Northern blot studies showed a straightforward relationship between class-I mRNA levels and H-2 expression. In vivo experiments demonstrated an inverse relationship between local growth and spontaneous metastasis, e.g., H-2-positive class-I clones produced high numbers of lung colonies but very poor local growth, and vice-versa. These results paralleled the NK sensitivity or resistance of the different clones. Cross-protection experiments showed that only clones coming from the same tumor were able to protect against challenge with clones of the same neoplasia but not with clones from different chemically induced fibrosarcomas, indicating that a clone of a given tumor probably contained the same TATA. Finally, we compared the H-2, K, D and L expression and class-I mRNA levels of various metastatic colonies. Interestingly, another degree of heterogeneity was found: an H-2-negative clone (GR9.B9) gave rise to H-2-negative (B9MP6) and H-2-positive (B9MP2) metastatic colonies.

The mechanisms of estrogen-induced cancer are still a matter of debate. Previous studies with stilbene estrogens and steroidal estrogens have shown that the in vitro transformation of primary Syrian hamster embryo (SHE) fibroblasts is a good experimental system for discriminating hormonal from nonhormonal events. We have now extended these studies to the triphenylethylene-type antiestrogens tamoxifen (TAM), 3-hydroxy-TAM, 4-hydroxy-TAM and 3,4-dihydroxy-TAM. Furthermore, a structural isomer of diethylstilbestrol (DES) with the hydroxy groups shifted into the meta-positions (3,3'-DES) was included. Our data clearly show that TAM and 4-hydroxy-TAM give rise to morphologic and neoplastic transformation of SHE cells in culture. In contrast, neither 3-hydroxy-TAM nor 3,3'-DES led to a significant transformation, in spite of the fact that both compounds are hormonally active. These results support the notion that nonhormonal events are essential for SHE cell transformation and suggest certain structural features to be necessary for the transforming capability. The putative carcinogenicity of TAM and 4-hydroxy-TAM but not of 3-hydroxy-TAM, as implied by these in vitro transformation studies, is corroborated by recent reports on ovarian and Leydig cell tumors in mice and hepatocellular carcinomas in rats.

SPARC (secreted protein acid and rich in cysteine), also known as osteonectin or BM-40, is a glycoprotein associated with the extracellular matrix of bone as well as with many soft tissues that produce extracellular matrix, including matrix-producing tumours. Northern and slot-blot analyses were used to study SPARC expression in tumours induced in vivo by methylcholanthrene (MCA) and in transformed cells induced in vitro by Kirsten-MSV and SV-40 infection. MCA-induced tumours expressed SPARC mRNA at quantitatively different levels. Fibroblasts transformed in vitro by Kirsten-MSV, and, to a lesser extent, by SV-40, showed reduced levels of SPARC mRNA expression compared with normal fibroblasts. Run-on assay indicated that transcription of SPARC was lower in the Kirsten-MSV transformed cells than in the normal parental fibroblast culture. However, SPARC mRNA in the transformed culture was as stable as that in normal culture. The difference, therefore, between levels of SPARC mRNA in transformed and normal culture was mainly due to different rates of transcription. Cloned cell lines derived from the Kirsten-MSV transformed culture also showed heterogeneous expression of SPARC: two lines had high and two had low expression of the gene. The level of mRNA correlated with that of the protein secreted. The SPARC expression might contribute to the malignant phenotype.

Macrophages (M phi) are multifunctional cells that regulate humoral and cellular immune responses. Our studies of tumor-induced M phi-mediated dysfunction used M phi subsets which were defined by their Mac-1, Mac-2, and Mac-3 surface markers. To measure the accessory activity of M phi for T cell alloreactivity, thioglycollate-elicited peritoneal M phi from normal and tumor-bearing hosts (TBH) were labeled with anti-Mac-1, -2, or -3 antibodies and separated by flow cytofluorometry. The separated Mac-1+, -2+, and -3+ M phi were called sorted M phi, while unseparated M phi were designated unsorted M phi. Both M phi types were added to mixed lymphocyte reaction (MLR) cultures at concentrations ranging from a low of 2% M phi to a high of 20% M phi. The low concentration of unsorted normal host M phi caused a 31% suppression of alloreactivity. Suppression reached 68% when high concentrations of unsorted normal host M phi were added to the MLR cultures. Unsorted TBH M phi reduced alloreactivity by 64% and 86% at low and high concentrations, respectively. When separated into subpopulations, normal host Mac-1+ M phi reduced alloreactivity by 48% and 81% when added at low and high concentrations, respectively. TBH Mac-1+ reduced alloreactivity by 31% and 59% at low and high concentrations, respectively. There were no differences in the suppression caused by normal or TBH Mac-2+ M phi, and by normal or TBH Mac-3+ M phi. Indomethacin treatment did not effect the suppression caused by Mac-1+ M phi, suggesting that proataglandin E2 was not involved. Indomethacin treatment did reduce suppression mediated by Mac-2+, -3+, and unsorted M phi. Mac-2+ M phi dramatically enhanced alloreactivity at low concentrations with normal host Mac-2+ M phi providing greater enhancement of alloreactivity than TBH Mac-2+ M phi. The division of M phi into subpopulations on the basis of Mac antigens suggested that Mac-1+ and -3+ M phi played a major role in immunosuppression in the normal host, while Mac-3+ M phi were more active in TBH immunosuppression. Because no one population of sorted TBH M phi was more suppressive than sorted normal host M phi, we suggest that tumor-induced immunosuppression may involve a network of suppressor M phi.

The c-Ha-ras-1 codon 61 A----T transversion is a highly consistent finding in murine skin DMBA-initiated/TPA-promoted papillomas and carcinomas. We performed Xba I RFLP analysis on DNA from clonal cell lines derived from 11 independent papillomas and a single carcinoma derived from topically applied DMBA and compared this to RFLP on DNA from 7 clonal fibrosarcoma cell lines derived from tumors arising after subcutaneous administration of the same carcinogen. The A----T point mutation was present in all of the papillomas and carcinoma derived by the classic DMBA-TPA protocol but was absent in all of the fibrosarcomas arising after subcutaneous administration of DMBA.

In addition to controlling the transition of resting normal cells from the G0-state of the cell cycle into S-phase, expression of the cellular protein p53 also seems to be necessary for the proliferation of cycling normal cells in an as yet undefined manner. To further elaborate the role of p53 in growing cells, we analysed p53 expression and its regulation in cells going into, and after release from, growth arrest at the restriction point (R-point) in the G1-phase of the cell cycle, induced by isoleucine depletion. Since growth arrest at the R-point is subject to internal control mechanisms of the cell cycle, this approach allowed us to include in our analyses normal Balb/c 3T3 fibroblasts, as well as cells of the chemically induced Balb/c fibrosarcoma cell line Meth A, expressing mutated p53. Isoleucine depletion induced a viable growth arrest at the R-point in cells of both cell lines, marked by a synchronous shut-down of DNA synthesis when the cells went into growth arrest, and a synchronous resumption of DNA synthesis after a lag period of about 2-4 h when the cells were released from growth arrest, as well as a shift to a G1 DNA content at the R-point. p53 expression in both cell lines showed a phenotypically similar regulation, as its synthesis was specifically reduced at the R-point. At the molecular level, however, p53 expression in growth arrested 3T3 cells was controlled at the transcriptional/post-transcriptional level, whereas control in growth arrested Meth A cells seemed to be at the level of mRNA translation. After release from growth arrest, p53 synthesis in both types of cells was rapidly restored, preceding resumption of total protein synthesis, and exhibiting a p53-specific profile.

To further characterize the role of p53 in growing normal Balb/c 3T3 fibroblasts, as well as of p53 in cells of the methylcholanthrene induced fibrosarcoma cell line Meth A, we analysed the effect of inhibition of p53 synthesis by microinjection of p53-specific monoclonal antibody PAb 122 into the nuclei of these cells after release from growth arrest induced by isoleucine starvation (see preceding paper [Steinmeyer et al., this issue] ). We show that microinjection of PAb 122, but not of control immunoglobulins, into the nuclei of both types of cells effectively blocked their re-entry into the S-phase of the cell cycle. Since isoleucine depletion of these cells was shown to lead to a growth arrest at the restriction point (R-point) in the G1-phase of the cell cycle, our results (i) define more precisely the role of p53 in growing cells as a protein controlling transition of the cells through this restriction point, and (ii) demonstrate that mutated p53 in Meth A cells still is functional with regard to cell cycle control at this restriction point. We suggest that p53 acts as a 'gate-keeping' protein at restriction points in the cell cycle, exerting a positive effect on the transition of cells through the cell cycle.

The role of neutrophils in transplantation immunity to syngeneic rat tumors was examined using a monoclonal antibody (RP-3) that depletes rat neutrophils selectively in vivo. We used 2 chemical carcinogen-induced transplanted tumors of different antigenic specificity (KMT-17 and KDH-8 of WKA rat origin). When neutrophils were selectively depleted by i.p. injection of RP-3 at the time of in vivo priming with X-irradiated tumor cells, the growth of subsequently s.c. transplanted identical tumors was not inhibited, in contrast to the group of rats immunized without RP-3 treatment. Tumor growth was also not inhibited when the immune rats were treated with RP-3 at the time of identical viable tumor cell challenge. These results suggest that neutrophils play a role in both the priming and effector phases of specific transplantation resistance to syngeneic tumors.

Ilmofosine is a cytostatic/cytotoxic thioether phospholipid derivative. The in vivo anti-tumour activity of this compound was investigated in a methylcholanthrene (MethA)-induced fibrosarcoma and in the 3Lewis-lung carcinoma systems, respectively. Ilmofosine showed antineoplastic and antimetastatic properties at oral doses ranging from 0.625 to 40 mg/kg/day. Combination of Ilmofosine (p.o.) together with either cyclophosphamide (p.o.) or cis-DDP (i.v.) resulted in synergistic effects in vivo. These results demonstrate the in vivo antitumour activity of Ilmofosine in two tumour systems. The data indicate that direct cytostatic/cytotoxic effects of Ilmofosine are mainly responsible for its antitumour activity in vivo and which are increased by other cytotoxics.

We have examined the metastatic capacity of different clones of a chemically induced fibrosarcoma GR9. These clones have previously been characterized for their H-2 class-I and class-II phenotype, NK sensitivity and local tumor growth. Our present data show that clones which express low amounts of H-2 class-I antigens are poorly metastatic in a post-surgical spontaneous metastasis assay, while those expressing high levels of class-I antigens possess a high metastatic capacity. These results correlate inversely with local growth patterns of the clones. High metastatic capacity was associated with resistance to NK cells. In an experimental metastasis assay, based on intravenous administration of in vitro carried GR9 clones to syngeneic BALB/c mice, an opposite result to the post-surgical assay was obtained. Gamma-IFN treatment of B9 clones (H-2-deficient) enhanced H-2 class-I expression and diminished experimentally induced metastases. Metastatic colonies, from the spontaneous metastasis assay, obtained from different organs, showed changes in the ratio H-2K/H-2D. There was a tendency for down-regulation of the expression of H-2K molecules in H-2-positive clones and for up-regulation of H-2D expression in H-2-negative clones.

Hematological values of the peripheral blood in the mice transplanted with methylcholanthrene-induced fibrosarcoma were examined. Values for red blood cells and platelets reduced after tumor transplantation. Values for white blood cells increased with the percentage of neutrophils being increased significantly. Phagocytic activity of neutrophils from the tumor-bearing mice was not reduced. The cultured fluid of the tumor used in the experiments possessed strong colony stimulating activity to bone marrow cells.

Immunity to carcinogen-induced transplantable fibrosarcomas (CHCT-NYU-4, -97, -36 and -20) in SC chickens was studied by the ability of spleen cells from NYU-4 or -97 immune chickens to proliferate in response to tumor cells in vitro. Spleen, but not peripheral blood cells, from NYU-4 immune chickens proliferated significantly more vigorously to gamma-irradiated NYU-4 cells than did cells from normal chickens. The proliferative response was not much affected by addition of indomethacin. Spleen cells from NYU-4-immune agammaglobulinemic (A gamma) chickens exhibit the same ability to proliferate in presence of gamma-irradiated NYU-4 tumor cells. Analysis of the phenotype of the T-cell component involved in proliferation showed that the proliferative response was significantly decreased by removal of CT4+ cells through indirect panning. Removal of CT8+ cells enhanced background proliferation without affecting the total thymidine incorporation in the presence of tumor cells. Immune spleen cells usually gave highest responses to the immunizing tumor, but also exhibited cross-reactivity to cells from other individual tumors induced by the same carcinogen.

Cryosurgery provides two prominent features, that is in situ destruction of malignant tumor and potential immunotherapy against tumor. In order to investigate the immunological response after cryosurgery, immunological system of macrophage activation were studied in an experimental tumor system using BALB/c mice and syngeneic Meth-A fibrosarcoma. The mice inoculated the cryodestroyed Meth-A (Cryo-Meth-A mice) acquired antitumor potency in Meth-A challenge test, and the mice inoculated the Mitomycin C treated Meth-A (MMC-Meth-A mice) also acquired antitumor potency. Spleen cells of Cryo-Meth-A mice showed antitumor activity in Winn assay, and spleen cells of MMC-Meth-A mice also showed antitumor activity. The effector cells in Cryo-Meth-A mice were mainly macrophages and natural killer (NK) cells. On the other hand, they were mainly macrophages and cytotoxic T lymphocytes (CTL) in MMC-Meth-A mice. The maximum macrophage cytostatic activity of Cryo-Meth-A mice was noted on 14 days after inoculation (Day 14), whereas it appeared on Day 7 and continued until Day 14 in MMC-Meth-A mice. Macrophages of Cryo-Meth-A mice enhanced the production activity of interleukin 1 (IL-1), interferon (IFN) and prostaglandin E2 (PGE2). On the other hand, macrophages of MMC-Meth-A mice enhanced production activity of tumor necrosis factor (TNF) and increased Ia antigen positive ratio. These findings suggest that macrophages of both groups are activated by different immunological mechanisms. It was found that Cryo-Meth-A played a role as an alpha-IFN inducer, and the macrophages stimulated with Cryo-Meth-A produced alpha-IFN in vitro. Intraperitoneal (i.p.) administration of the anti-alpha-IFN antibody carried down-regulation of macrophage cytostatic activity and NK activity of Cryo-Meth-A mice, whereas MMC-Meth-A mice were not. In addition to these findings, CTL activity enhanced some extent by i.p. administration of the anti-alpha-IFN antibody. These facts suggest that the alpha-IFN have two ways of immunological regulation, the first one is the activation of macrophage cytostatic activity and NK activity, and the other is down-regulation of CTL activity by suppression of Ia antigen expression of macrophages. These results suggest that cryodestruction changes the tumor antigen of Meth-A cells, thereby Cryo-Meth-A induces peculiar immunological response.

The two types of transplantable methyl cholanthrene induced fibrosarcoma in rats was used to find out the possible relationship between the fibrinolytic, procoagulant activities and the metastasizing capacity of the tumours. The highly metastatic tumour seems to possess high fibrinolytic activity as compared to the low metastatic one. Interestingly enough, it was found that the procoagulant and fibrinolytic activities in the highly metastasising tumour bear inverse relationship with each other in relation to time of tumour growth. The procoagulant activity of the tumour bypasses factor VII and acts at factor X level. The plasminogen activator present in the tumour tissue has been characterized by sephadex G-200 column chromatography and PAGE.

In vivo 31P nuclear magnetic resonance (31P NMR) spectroscopy has been used to compare metabolic profiles with tumor radiosensitivity. A radioresistant mammary carcinoma (MCa) and a radiosensitive methylcholanthrene-induced fibrosarcoma (Meth-A) were studied by 31P NMR spectroscopy in the tumor volume range of approximately 100-1200 mm3. The MCa showed a constant pH in this volume range; the ratio of phosphocreatine to inorganic phosphate (PCr/Pi) for 160-300 mm3 tumors was 0.33 +/- 0.11 (mean +/- standard deviation) and did not change (0.29 +/- .09) for tumors in the volume range of 600-1200 mm3. In comparison, the Meth-A showed a decrease in tumor pH as volume increased from 160-300 mm3 (pH 7.16 +/- 0.4) to 600-1200 mm3 (pH 6.94 +/- .07). Tumor PCr/Pi decreased from 0.70 +/- .16 (160-300 mm3) to 0.33 +/- .16 (600-1200 mm3). The radiation doses for control of MCa-induced tumors in 50% of the treated tumors ranged from 65 (150-250 mm3) to 71 Gy (1000-1300 mm3) and for the Meth-A-induced tumors ranged from 35 (150-250 mm3) to 38 Gy (1000-1300 mm3). These results suggest that 31P NMR spectra may be a qualitative predictor of tumor hypoxia, although further studies of human and rodent tumors are necessary to support this hypothesis.

Recombinant murine tumor necrosis factor (TNF) was investigated for its ability to increase the response of a murine mammary carcinoma to fractionated local tumor irradiation. When tumors in the leg of syngeneic mice grew to 8 mm in diameter (268 mm3), they were exposed to 3, 4 or 5 Gy gamma-rays daily for 10 days. Tumor necrosis factor was given 3 hr after each irradiation at a dose of 2 micrograms per mouse. Tumor growth delay was used as the endpoint. The effect of treatment with both agents combined was greater than the additive effect of the individual treatments. Tumor necrosis factor increased local tumor radiotherapy response by a factor of about 1.2, and by itself was effective in retarding the growth of both immunogenic and nonimmunogenic tumors. These experiments suggest that tumor necrosis factor in combination with radiotherapy may be beneficial for the treatment of cancer patients.

We examined the effect of allogeneic blood transfusions (BT) on pulmonary metastases in a mouse model. Recipient (C57B1/6J) mice were transfused with either saline, syngeneic blood or allogeneic (Balb/c) blood on two occasions, days 0 and 3. One week after the last transfusion, recipient mice were injected intravenously with varying numbers of methylcholanthrene-induced fibrosarcoma cells. Twenty days later the number of pleural nodules was counted as an index of pulmonary metastasis. The data demonstrate that the inoculation of 2.5 x 10(5) or 1 x 10(5) tumor cells resulted in significantly higher numbers of pulmonary metastases in mice that received allogeneic BT than the mice that received syngeneic blood or saline. In contrast, allogeneic BT caused significant inhibition of pulmonary metastases in mice that received 3.5 x 10(5) tumor cells. The data suggest that the immunomodulatory (stimulatory or inhibitory) effect of BT is dependent on the numbers of tumor cells inoculated. It is likely that the conflicting reports in the literature on the effects of BT on tumor growth may be due to inoculation of different numbers of tumor cells. These results have an important bearing in understanding the effect of allogeneic BT on tumor growth both in experimental animals and in cancer patients.