The importance of p53-independent apoptosis in the intestinal toxicity induced by raltitrexed (ZD1694, Tomudex): genetic differences between BALB/c and DBA/2 mice.

The thymidylate synthase inhibitor raltitrexed (ZD1694, Tomudex) induces greater intestinal toxicity, manifested as diarrhea and weight loss, in BALB/c than in DBA/2 mice. No convincing pharmacokinetic or pharmacodynamic reason for this strain difference has been established. We have investigated whether this strain difference in response to raltitrexed is related to differential susceptibilities of intestinal mucosae to undergo apoptosis and also whether p53 expression, a critical factor in 5-fluorouracil-induced intestinal apoptosis and toxicity, modulates this response. Ten mg/kg or 100 mg/kg raltitrexed were administered as single or double i.p. injections 24 h apart to BALB/c, DBA/2, and p53-/- mice. Apoptosis, mitosis, and tissue damage were assessed in intestinal epithelium, and animal weight was recorded. BALB/c mice developed diarrhea and weight loss following 100 mg/kg x2 raltitrexed, whereas DBA/2 mice did not. BALB/c mice were more sensitive than DBA/2 to induction of small-intestinal and colonic apoptosis 24 h following 100 mg/kg raltitrexed. Inhibition of mitosis was equivalent in both strains. Both strains showed histopathological damage to the small intestine after 100 mg/kg x2 raltitrexed, but only BALB/c mice demonstrated colonic damage. p53-null mice showed the same level of small intestinal apoptosis as their wild-type counterparts 24 h after 100 mg/kg x1 raltitrexed and also the same levels of intestinal toxicity 3, 5, and 7 days after 100 mg/kg x2 raltitrexed. Thus, BALB/c mice were more susceptible to induction of intestinal apoptosis by raltitrexed than DBA/2 mice and also demonstrated more histopathological damage in the colon correlating with the induction of diarrhea and weight loss. In contrast to 5-fluorouracil, the intestinal apoptosis and toxicity induced by raltitrexed were p53-independent.     

DBA/2-like minor histocompatibility antigens on a BALB/c lymphoma. A BALB/c anti-DBA/2 serum which lyses the tumor and blocks BALB/c anti-tumor and anti-DBA/2 effectors

We have previously shown that BALB/c anti-DBA/2 T cells can lyse the Moloney virus-induced BALB/c lymphoma YC8. In order to determine whether serologically defined minor histocompatibility antigens (MiHA) cross-reacting with those of DBA/2 tissues are present on YC8, we produced an antiserum directed against non-H-2 antigens by immunizing BALB/c mice with DBA/2 Con A and lipopolysaccharide (LPS)-induced lymphoblasts. In a direct complement-dependent cytotoxicity assay, the antiserum (OR-1) lysed DBA/2 and YC8 but not BALB/c lymphocytes and blasts. No reactions against viral antigens were detected in the antisera as shown by the lack of cytotoxicity on a panel of lymphomas expressing a variety of viral antigens. In addition, OR-1 was able to specifically block a cytotoxic T lymphocyte (CTL), H-2-restricted BALB/c anti-DBA/2 cytotoxic response when bound to DBA/2 or to YC8 target cells. These results indicate that antigens cross-reacting between YC8 lymphoma and DBA/2 tissues are serologically defined MiHA of DBA/2 background and that OR-1 serum can block a CTL reaction by binding to target antigen rather than to major histocompatibility complex products.     

Distribution, covalent binding, and DNA adduct formation of 7,12-dimethylbenz(a)anthracene in SENCAR and BALB/c mice following topical and oral administration

SENCAR mice are much more susceptible to tumor initiation by 7,12-dimethylbenz(a)anthracene (DMBA) administered topically than p.o. and are also more susceptible to initiation by topically applied DMBA than are BALB/c mice. To determine how the distribution and metabolic activation of DMBA differed in these strains and with route of administration, BALB/c and SENCAR mice were exposed to [3H]DMBA topically and p.o., and the distribution and DNA binding of DMBA were analyzed. Both the amount of DMBA in skin and the covalent binding of DMBA to epidermal DNA were greater following topical administration than after p.o. administration in both strains. Differences in DMBA distribution and macromolecular binding were found between SENCAR and BALB/c mice, with the binding of DMBA to DNA in epidermis tending to be greater in BALB/c mice than in SENCAR mice when differences were observed. The formation of individual DMBA:DNA adducts in epidermis was also examined in SENCAR and BALB/c mice following topical administration of DMBA. No substantial qualitative or quantitative differences in DMBA:DNA adducts were found between SENCAR and BALB/c mice. The anti/syn-DMBA-diol-epoxide-DNA adduct ratios calculated from the three major DMBA:deoxyribonucleoside adducts increased with time in both SENCAR and BALB/c mice. The data suggest that differences in the distribution and macromolecular binding of DMBA are responsible for the much greater skin tumor initiating activity of DMBA applied topically than p.o. but do not account for the greater sensitivity of the SENCAR mouse to DMBA-induced epidermal tumorigenesis.     

Alloreactivity and tumor antigens: generation of syngeneic antilymphoma killer lymphocytes by alloimmunization of mice with normal cells

The possibility of obtaining a syngeneic antitumor effect by immunization with normal allogeneic cells was investigated by tests of the lytic activity of peritoneal exudate cells (PEC) of BALB/c mice immunized with lymphoid cells of either a single strain or a pool of six different allogeneic strains on the syngeneic Moloney virus-induced lymphoma YC8 target and on one of its clones designated YC8-D1. Significant cytotoxicity on both targets but not on two other BALB/c lymphomas was obtained with PEC of BALB/c mice singly immunized to the non-H-2-incompatible but H-2-compatible DBA/2 or B10.D2 lymphoid cells. The lack of lysis of YC8 cells by PEC of BALB/c mice immune to B10.A (H-2k,d) suggests that the in vitro killing was restricted by Kd-IEd region products of the major histocompatibility complex. Pool immunization was effective in generating antitumor cytotoxic lymphocytes only when DBA/2 lymphoid cells were included in the pool. The pattern of reactivity of effectors elicited in (BALB/c x DBA/2)F1 and in (BALB/c x B10.D2)F1 mice by immunization with DBA/2 and B10.D2 cells showed that at least two sets of antigens are recognized on YC8 targets, one shared by DBA/2 and B10.D2 tissues and the other expressed by DBA/2 cells only. Cold target blocking experiments indicated that the same effectors recognized non-H-2 antigens of DBA/2 and the cross-reacting YC8 determinants. The antitumor effect was mediated by T-cells, since it was abrogated by treatment of effectors with anti-Thy 1.2 serum plus complement. These data indicate that determinants defined by cytotoxic T-lymphocytes are expressed on the BALB/c lymphoma YC8 and cross-react with non-H-2 antigens of DBA/2 and B10.D2 strains.     

Immunological responses to a murine mammary adenocarcinoma. Outgrowth of pulmonary occult metastases induced by immunosuppressive perturbations

Intravenously (i.v.) or subcutaneously (s.c.) injected T1699 tumor cell subpopulations (Ts, and TLI-1) were destroyed more rapidly in BALB/c nu/nu athymic mice than in syngeneic DBA/2J mice. The number of artificial pulmonary metastases of TLI-1 tumors was significantly lower, and the growth rate of s.c. Ts, and TLI-1 tumors was correspondingly slower in the nude mice. No macroscopic outgrowth of pulmonary metastases of Ts tumors was detected in either group of s.c. tumor-bearers, even though the s.c. tumors progressed and killed the hosts. Unexpectedly, however, s.c. implantation in normal DBA/2J mice of lung homogenates not only from DBA/2J but also from s.c. Ts tumor-bearing BALB/c nu/nu mice produced tumors, suggesting that significant numbers of clonogenic Ts cells were present in the lungs of animals without spontaneous outgrowth of pulmonary metastases. Perturbations of s.c. Ts tumor-bearing DBA/2J or BALB/c nu/nu mice with immunosuppressive drugs or with anti-mouse thymocyte serum, respectively, induced rapid outgrowth of pulmonary metastases.     

Suppressor macrophages in tumor-bearing mice. Inconsistency between in vivo and in vitro findings?

The growth of a spontaneous adenocarcinoma (ADK-1t) of BALB/c mice (H-2d, MIsb) induces progressive hyporesponsiveness of spleen cells stimulated with PHA or irradiated leukocytes from DBA/2 strain (H-2d, MIsa) in mixed leukocyte culture. This hyporesponsiveness is due to the suppressor activity of macrophage-like cells, since it is abrogated by passage over nylon-wool columns, pretreatment with carrageenans, or carbonyl iron and magnet. BALB/c mice bearing greater than 20 mm mean diameter ADK-1t tumors, and displaying a drastic impairment of T lymphocyte reactivity in vitro, were challenged with the P815 mastocytoma of the DBA/2 strain. These mice rejected increasing numbers of P815 cells to the same extent as normal BALB/c mice. The reactivity towards P815 is abrogated by 450 rad, and can be reconstituted by the adoptive transfer of 2 X 10(7) normal T lymphocytes in either normal or ADK-1t bearing BALB/c mice. These data show that a discrepancy exists between the capacity of T lymphocytes from ADK-1t bearing mice to react against DBA/2 cells tested in vitro or in vivo, and indicates that great caution should be exercised in predicting the responsiveness of the immune system on the basis of in vitro data on the suppressor activity of macrophages.     

Adoptive chemoimmunotherapy of a moloney lymphoma

Adult BALB/c mice were inoculated with BALB/c Moloney lymphoma cells. On day 6, mice with palpable tumors received sublethal cyclophosphamide (CY) and BALB/c or DBA 2 spleen cells. All untreated mice died with tumor on days 8-12. Spleen cells alone had no effect. All mice treated with CY alone died with tumor by day 30, as did all mice treated with CY plus cells from non-immune donors or from DBA donors immunized against normal BALB/c tissue. By contrast, treatment with CY plus cells from BALB/c or DBA/2 donors immunized with antigenically-related murine sarcoma virus (Moloney) was moderately effective — 30 of 65 mice lived beyond day 60 and 14 of 65 beyond day 100. Lethal X-irradiation of the immune cells abolished their efficacy. Thus, an established antigenic lymphoma was inhibited or eradicated by a combination of chemotherapy and immunotherapy with spleen cells from syngeneic or H-2-compatible donors, but only if the cells were viable and were derived from donors pre-immunized against tumor-associated antigens. Finally, when lethal X-irradiation was substituted for sublethal CY, immunotherapy was not effective, probably because the tumor cells were far less sensitive to X-ray than to CY.     

Strain-dependent lung tumor formation in mice transplacentally exposed to 3-methylcholanthrene and post-natally exposed to butylated hydroxytoluene.

The carcinogenic effects of in utero exposure to 3-methylcholanthrene (MC) have been demonstrated in the tumor-resistant C57BL/6 (B6) and DBA (D2) strains of mice. In this study, we determined the effects of in utero exposure to MC in BALB/c mice, a strain which demonstrates greater susceptibility to lung tumor induction, and compared our findings with those previously found in [D2xB6D2F(1)]F(2) mice. In addition, we assessed the molecular pathogenesis of the chemically induced tumors and examined the effects of the putative lung tumor promoter butylated hydroxytoluene (BHT) in BALB/c mice. BALB/c mice were treated on day 17 of gestation with 5, 15 or 45 mg/kg MC and 6 weeks after birth with BHT for 6 consecutive weeks. Mice were killed at 6 months of age. Ki-ras, p16Ink4a and p19ARF gene loci were amplified from paraffin-embedded lung tumor tissue and screened for the presence of point mutations via allele-specific oligonucleotide hybridization and single strand conformation polymorphism (SSCP) analyses. Ki-ras point mutations were found in 56% (20/36) of BALB/c lung tumors, with 33% (2/6) of the hyperplasias, 58% (10/19) of the adenomas and 73% (8/11) of the carcinomas exhibiting point mutations at this gene locus. Similar incidences of Ki-ras mutations were previously found following transplacental exposure of [D2xB6D2F(1)]F(2) mice to MC and treatment of adult A/J mice with urethane. Interestingly, a strain-dependent difference was observed in the mutational spectrum. Sixty-two and 38% of the lung lesions in BALB/c mice exhibited G-->C and G-->T transversions, respectively, in contrast to the 13 and 84% incidences previously observed in [D2xB6D2F(1)]F(2) mice. SSCP analysis of the tumor suppressor gene p16Ink4a showed a 6% incidence of point mutations, consistent with that found in [D2xB6D2F(1)]F(2) mice. No mutations were found in exon 1beta of the p19ARF gene of either strain. BHT, a lung tumor promoter in adult mice, had no statistically significant effects on either tumor incidence, tumor multiplicity or the mutational spectrum produced in the Ki-ras gene by in utero MC treatment. However, though not significant, there was an observable trend in increased tumor multiplicity in mice co-treated with BHT. These data demonstrate the transplacental carcinogenic effect of MC in BALB/c mice and show that mutagenic damage to Ki-ras is a critical early event mediating murine lung tumorigenesis in both the tumor-sensitive and tumor-resistant strains. Unlike what occurs when adult BALB/c mice are treated with MC, BHT does not appear to significantly promote the formation of lung tumors following transplacental exposure to MC, possibly due to the rapid growth and cell proliferation in the developing organism. Strain-dependent differences in the Ki-ras mutational spectrum may be associated with their differential susceptibility to lung tumor initiation.     

Elevated breast cancer risk in irradiated BALB/c mice associates with unique functional polymorphism of the Prkdc (DNA-dependent protein kinase catalytic subunit) gene

Female BALB/c mice are unusually radiosensitive and more susceptible than C57BL/6 and other tested inbred mice to ionizing radiation (IR)-induced mammary tumors. This breast cancer susceptibility is correlated with elevated susceptibility for mammary cell transformation and genomic instability following irradiation. In this study, we report the identification of two BALB/c strain-specific polymorphisms in the coding region of Prkdc, the gene encoding the DNA-dependent protein kinase catalytic subunit, which is known to be involved in DNA double-stranded break repair and post-IR signal transduction. First, we identified an A --> G transition at base 11530 resulting in a Met --> Val conversion at codon 3844 (M3844V) in the phosphatidylinositol 3-kinase domain upstream of the scid mutation (Y4046X). Second, we identified a C --> T transition at base 6418 resulting in an Arg --> Cys conversion at codon 2140 (R2140C) downstream of the putative leucine zipper domain. This unique PrkdcBALB variant gene is shown to be associated with decreased DNA-dependent protein kinase catalytic subunit activity and with increased susceptibility to IR-induced genomic instability in primary mammary epithelial cells. The data provide the first evidence that naturally arising allelic variation in a mouse DNA damage response gene may associate with IR response and breast cancer risk.     

Inhibition of spontaneous and experimental metastasis by a new derivative of camptothecin,CPT-11, in mice

Summary The antimetastatic effect of a new water-soluble derivative of camptothecin, 7-ethyl-10-(4-(1-piperidino)-1-piperidino) carbonyloxy-camptothecin (CPT-11), were examined in several metastatic murine tumor systems. Intravenous (i.v.) injection of CPT-11 into BALB/c mice inhibited lung metastasis by i.v. inoculated, metastatic colonic adenocarcinoma 26 (C26) cells, C26NL-17, in BALB/c mice. This treatment was also effective in C57BL/6 mice against lung metastasis by i.v. inoculated B16-F10 and B16-BL6 cells, highly metastatic variants of the B16 melanoma. Furthermore, intraperitoneal (i.p.) injection of CPT-11 significantly inhibited the growth of C26NL-22 cells, a highly metastatic variant of C26, inoculated subcutaneously (s.c.) into the left front footpads of BALB/c mice. Also, i.p. or i.v. injection of CPT-11 effectively inhibited the growth of 3LL tumors inoculated s.c. into the hind footpads of C57BL/6 mice. Moreover, following s.c. inoculation of either C26NL-22 or 3LL cells, combined surgical excision of the primary tumor and either i.p. or i. v. CPT-11 injections given before or after surgery markedly inhibited the formation of pulmonary metastases. These results show that a new derivative of camptothecin, CPT-11, has a potent inhibitory effect against both spontaneous and experimental lung metastasis.     

Differential susceptibility of BALB/c and DBA/2 cells to plasmacytoma induction in reciprocal chimeras

Reciprocal chimeras were generated between BALB/c and DBA/2 mice by inoculating newborn recipients of either strain with bone-marrow (BM) cells of the other through the periorbital vein. DBA/2 mice inoculated with the BALB/c with proven chimerism will be referred to as C----D, the reciprocal as D----C. The BALB/c cells carried a Robertsonian 6;15 (Rb6;15) chromosome marker to facilitate identification. The chimeric mice contained between 5% and 70% of donor cells when examined at 4 to 5 weeks of age. Six of 10 C----D developed plasmacytomas (MPC) after 3 x 0.5 ml monthly pristane treatment (incidence 60%) and 8 of 25 (incidence 32%) after 2 to 3 x 0.5 ml pristane followed by Abelson virus (A-MuLV) infection. Seven of 15 D----C developed MPC after pristane treatment (incidence 47%) and 4 of 17 after pristane + A-MuLV (incidence 24%). All tumors that have arisen in both reciprocal chimeras originated from BALB/c cells independently of the degree of chimerism. All tumors contained an Ig/myc translocation. Among the C----D chimeras, 5 carried t(12;15) and I t(6;15) in the pristane-treated group, while 4 carried t(12;15), I t(6;15) and 3 t(15;16) in the pristane + A-MuLV. Among the D----C chimeras 6 carried t(12;15) and I t(6;15) in the pristane-treated group, while 3 t(12;15) and I t(6;15) in the pristane + A-MuLV. No tumors developed in 18 pristane- and 22 pristane + A-MuLV-treated DBA/2 mice nor in 15 pristane- and 17 pristane + A-MuLV-treated (BALB/c x DBA/2)F1 mice. The data indicate that BALB/c and DBA/2 cells differ in their propensity to transform into plasmacytoma in identical host environments after both pristane and pristane + A-MuLV treatment. They also show that the oil granuloma can support MPC development in either type of chimeric host.     

Selective depletion of nk cell activity in vivo and its effect on the growth of NK-sensitive and nk-resistant tumor cell variants

Intravenous injection of rabbit anti-asialo-GM₁ serum, an antiserum previously shown to eliminate splenic natural killer (NK) activity in vitro, profoundly depressed NK activity in CBA, DBA/2 and BALB/c nu/nu mice. The effect on NK activity was selective, as treatment of mice with anti-asialo-GM₁ serum did not affect the development of other cytotoxic cells including cytotoxic macrophages following injection of poly I:C, or cytotoxic T cells in response to allogeneic cells. The role of NK cells in controlling tumor cell growth was investigated using an NK-sensitive (cl 27v-IC2) and an NK-resistant (cl 27av) subline of the murine lymphoma L5178Y. Initial studies showed that cl 27v-IC2 cells were at least 100 times less tumorigenic than were cl 27av cells in both syngeneic DBA/2 mice and BALB/c nu/nu mice. In addition, treatment of DBA/2 mice with poly I:C, which boosted NK activity, markedly depressed the growth of cl 27v-IC2 cells, but not of cl 27av cells. On the other hand, treatment of DBA/2 mice and BALB/c nu/nu mice with antiasialo-GM1₁ serum led to a marked increase in tumorigenicity of cl 27v-IC2 cells, but had no effect on the tumorigenicity of cl 27av cells. In addition, the protection against cl 27v-IC2 growth afforded by poly-I:C treatment was abrogated by injection of anti-asialo-GM₁ serum. The possibility that the effects observed were caused by binding of the injected antibodies to the tumor cells was minimized by: (1) using a clone of tumor cells (cl 27v-IC2) that lacks chemically detectable asialo-GM₁, and (2) pre treating animals with anti-asialo-GM₁ rather than administering antiserum and tumor cells concurrently. These studies provided compelling evidence that NK cells could play an active role in controlling tumor growth. Selective depletion of NK activity by injection of antiasialo-GM₁ serum is a method which would be generally applicable to studying the role of NK cells in disease processes.     

Significance of transplacental hemorrhage in the induction of specific maternal unresponsiveness

Transplacental hemorrhage is reportedly detectable by erythrocytes containing fetal hemoglobin-F in the maternal circulation. The procedure in the present study is based on the premise that fetal hemoglobin is not eluted from red cells in blood smears by an acid buffer, whereas adult hemoglobin is removed. Erythrocytes containing hemoglobin-F were observed in blood smears of both virgin and pregnant BALB/c mice, but the relative number of these cells in the maternal blood increased during pregnancy, reached a peak around the time of parturition, and decreased thereafter. In BALB/c females pregnant by DBA/2 males, the relatively large numbers of acid-resistant erythrocytes in maternal blood were related to the maternal unresponsiveness to DBA/2 allografts. The fetal derivation of at least a fraction of these cells was suggested by the apparent activity of fetal immunogens in the maternal circulation. The blood of peripartum BALB/c mice multiparous by DBA/2 males was injected into normal, nonimmune BALB/c mice. The blood recipients were later challenged with an allotransplantable DBA/2 tumor. A low degree of immunity was indicated by a decreased size of tumor implants. Attempts to demonstrate vertical transmission of immune suppression from mothers to progeny were negative. These findings indicate that transplacental hemorrhage is a potential source of antigens involved in the induction of specific maternal unresponsiveness.     

Immune-mediated arrest and reversal of established visceral metastases in athymic mice.

The metastasizing MDAY-D2 tumor of DBA/2 mice disseminates in BALB/c allogeneic athymic nude (nu/nu) mice in a manner identical to that observed in the syngeneic host. Both the kinetics and organ distribution pattern of metastases from s.c. implants of MDAY-D2 are routinely predictable at any given tumor dose. BALB/c heterozygote (nu/+) litter-mates reject MDAY-D2 grafts on the basis of the multiple minor histocompatibility differences that exist between DBA/2 and BALB/c mice. The in vitro cell-mediated cytotoxic response detected in tumor-bearing BALB/c nu/+ mice is "low grade" (isotope release is approximately 40 to 50% by 24-hr 111-indium-8-hydroxyquinoline assay and approximately 6 to 8% by 6-hr 51Cr assay) and yet correlates directly with tumor rejection. BALB/c nu/nu mice can be protected against MDAY-D2 by previous reconstitution with lymphoid cells from normal or MDAY-D2-sensitized BALB/c nu/+ mice. In addition, surgically documented, established visceral metastases in BALB/c nu/nu mice can be arrested and regressed by the adoptive transfer of MDAY-D2-sensitized BALB/c nu/+ spleen cells. This represents one of the few models where established metastases have been immunotherapeutically regressed. As such, the MDAY-D2 BALB/c nu/nu mouse model offers unique advantages for studying the role of the immune system in regulating the metastatic process.     

Changes of serum thymic factor levels in Friend leukemia virus-infected mice.

Sera and spleen cells from DBA/2 and BALB/c mice were sampled at various time intervals after infection with Friend leukemia virus (FLV, polycythemic strain) and assayed for the presence of serum thymic factor (STF) and theta-positive cells, respectively.A pronounced and dose-dependent decrease of STF levels was observed in DBA/2 mice as early as 48 h after infection; STF levels were decreased in BALB/c mice, too, but the effect was less marked and long-lived than in DBA/2 mice.In close parallelism to the fall of STF levels in the sera, theta-positive cells were also found to be markedly decreased in the spleens of DBA/2 mice and, to a lesser extent, in BALB/c mice. Thetapositive cells were assayed by measuring the sensitivity to azathioprine of spleen rosette-forming cells.Both the fall of STF levels and the decrease of theta-positive spleen cells do not persist longer than 4 weeks and are not, therefore, related to the state of overt leukemia.Conceivably, though, these changes, that appear to be virus-related, may influence the early fate of FLV-transformed cells by lowering some thymus-dependent functions which may be relevant to the immunological surveillance.     

Immunogenicity of solubilized tumor antigen extracted from P1798 murine lymphoma cells or isolated from tumor-bearer ascites fluid and reactivity with anti-thy-1.2 antiserum.

Solubilized antigen was prepared from P1798 lymphoma cells by sonication, 3 M KCI extraction, or isolated from the ascites fluid of syngeneic tumor-bearing BALB/c mice. Antigen was detected and quantitated by its ability to block activity of anti-P1798 serum raised in syngeneic mice, as assayed by cytotoxic and indirect immunofluorescence tests. It was established that the reaction was immunologically specific as the P1798 antigen did not inhibit the binding to L1210 lymphoma cells of antisera raised against L1210 in syngeneic DBA/2 or allogeneic BALB/c mice. Vaccination of BALB/c mice with different subcellular fractions of sonicated antigen or with ascites fluid resulted in protection against a live P1798 challenge with results comparable to those obtained using iodoacetamide-modified tumor cells. Solubilized antigen prepared by each of the three methods eluted from a Bio-Gel A5m agarose column exclusively in an early peak that had a molecular weight estimated to be greater than 2 X 10(6). This column-fractionated antigen was shown to cross-react with antiserum raised against Thy-1.2 antigen, which is present on P1798 cells. The purified P1798 antigen sedimented at 200,000 g and was shown to protect syngeneic mice in immunoprophylactic tests.     

Alien histocompatibility determinants on the cell surface of sarcomas induced by methylcholanthrene. I. In vivo studies.

Groups of BALB/c mice were immunized to normal tissues (skin and/or liver plus kidney) of C3Hf, C57Bl/6, DBA/2 and AKR strains and challenged with either of two syngeneic 3-methylcholanthrene-induced immunogenic sarcomas, ST2 and TZ15, or with a "spontaneous" non-immunogenic BALB/c sarcoma, B2. It was found that anti-C3Hf and anti-DBA/2 immune mice were significantly protected against the growth of ST2, whereas anti-AKR immune mice rejected TZ15; no protection was elicited by immunizing with normal tissues of any strain against B2, which lacked individual tumor-associated transplantation antigens (TATA). The reciprocal experiment, i.e. the immunization of BALB/c mice with tumor cells and challenge with skin grafts of different strains, was also carried out with ST2 and TZ15. Accelerated rejection of all the various allogeneic skins was observed in anti-ST2 immune mice and of AKR and C3Hf skin in anti-TZ15 immune animals. In addition the Winn test demonstrated that lymph-node cells of BALB/c mice immune to C3Hf or DBA/2 tissues were specifically inhibitory for ST2, and that lymph-node cells immune to AKR tissues protected against TZ15. In a further experiment both ST2 and TZ15 tumors were left to grow in (C3Hf X BALB/c)F1, (C57Bl/6 X BALB/c)F1, (BALB/c X DBA/2)F1 and (BALB/c X AKR)F1 mice; the tumors were then excised and the "immune" mice challenged with the related tumor to measure their immune response in comparison with that elicited by the same procedure in BALB/c mice. ST2 was highly immunogenic in syngeneic BALB/c mice and in all the hybrid combinations except (C3Hf X BALB/c)F1 mice, where it completely lost its immunogenicity; TZ15 showed a certain loss of immunogenic strength in (BALB/c X AKR)F1 hybrids. It was concluded that TATA of ST2 contain antigenic determinants expressed on the normal cells of C3Hf and DBA/2 strains, and that TATA of TZ15 are likely to share antigens with AKR normal tissues.     

Ecotropic MuLV expression in radiation-induced lymphomas of the RF, BALB/c and (BALB/c X RF)F1 mouse strains

Endogenous ecotropic viruses isolated from radiation-induced lymphomatous tissue of BALB/c mice have been shown to consist of a collection of variants as assayed by the mobility of virion structural proteins p30, p15, p12 and gp70 on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Ellis et al., 1980 a). In this study we show that a similar phenomenon occurs in RF mice, but only with regard to p15 and gp70, and not p30 and p12. Using the distinct and unvarying mobility of the RF viral p12 protein on SDS-PAGE as a marker, we show that the RF-derived ecotropic murine leukemia virus (MuLV) is expressed to the exclusion of the BALB/c-derived ecotropic MuLV in F1 hybrid mice of the BALB/c X RF cross, and that variant viruses expressed in F1 radiation-induced lymphomatous tissue display the pattern of variation characteristic of the RF, and not of the BALB/c, strain.     

Ionizing radiation enhances immunogenicity of cells expressing a tumor-specific T-cell epitope.

BACKGROUND: p53 point mutations represent potential tumor-specific cytolytic T lymphocyte (CTL) epitopes. Whether ionizing radiation (IR) alters the immunological properties of cells expressing mutant p53 in respect of the CTL epitope generated by a defined point mutation has not been evaluated. METHODS: Mutant p53-expressing syngeneic, nontumor forming BALB/c 3T3 fibroblasts, tumor forming ras-transfected BALB/c 3T3 sarcomas, and DBA/2-derived P815 mastocytoma cells, which differ at the level of minor histocompatibility antigens, were used as cellular vaccines. Cells were either injected with or without prior IR into naive BALB/c mice. Cellular cytotoxicity was assessed after secondary restimulation of effector spleen cells in vitro. RESULTS: Injection of P815 mastocytoma cells expressing the mutant p53 induced mutation-specific CTL in BALB/c mice irrespective of prior irradiation. However, syngeneic fibroblasts or fibrosarcomas endogenously expressing mutant p53 were able to induce significant mutation-specific CTL only when irradiated prior to injection into BALB/c mice. IR of fibroblasts did not detectably alter the expression of cell surface molecules involved in immune response induction, nor did it alter the short-term in vitro viability of the fibroblasts. Interestingly, radioactively-labeled fibroblasts injected into mice after irradiation showed altered organ distribution, suggesting that the in vivo fate of these cells may play a crucial role in their immunogenicity. CONCLUSIONS: These findings indicate that IR can alter the immunogenicity of syngeneic normal as well as tumor forming fibroblasts in vivo, and support the view that ionizing radiation enhances immunogenicity of cellular tumor vaccines.     

Identification of a region of homozygous deletion on 8p22-23.1 in medulloblastoma

To identify critical tumor suppressor loci that are associated with the development of medulloblastoma, we performed a comprehensive genome-wide allelotype analysis in a series of 12 medulloblastomas. Non-random allelic imbalances were identified on chromosomes 7q (58.3%), 8p (66.7%), 16q (58.3%), 17p (58.3%) and 17q (66.7%). Comparative genomic hybridization analysis confirmed that allelic imbalances on 8p, 16q and 17p were due to loss of genetic materials. Finer deletion mapping in an expanded series of 23 medulloblastomas localized the common deletion region on 8p to an interval of 18.14 cM on 8p22-23.2. We then searched within the region of loss on 8p for loci that might contain homozygous deletion using comparative duplex PCR. An overlapping homozygous deletion region was identified in a 1.8 cM interval on 8p22-23.1, between markers D8S520 and D8S1130, in two medulloblastomas. This region of homozygous deletion also encompasses the 1.4 cM minimal deletion region detected on 8p in ductal carcinoma in situ of breast. In conclusion, we reported for the first time a detailed deletion mapping on 8p in medulloblastoma and have identified a region of homozygous deletion on 8p22-23.1 that is likely to contain a critical tumor suppressor gene involved in the development of medulloblastoma.