BCG inhibition of murine leudemia: local suppression and systemic tumor immunity require different doses.

The quantitative relationship between bacillus Calmette-Guérin (BCG) and tumor cells which are optimal for suppressing the growth of tumor cells in BCG-tumor cell mixtures are detrimental to the development of a sustained, systemic tumor rejection immunity in the LSTRA murine leukemia.

Genetically obese mice: resistance to metastasis of B16 melanoma and enhanced T-lymphocyte mitogenic responses.

The metastasis of B16 melanoma cells differed significantly in obese (ob/ob) and lean (+/?) female mice of strain C57BL/6J. When the mice were inoculated subcutaneously with melanoma cells at 10 to 11 months of age, the primary tumor grew more slowly in obese than in lean littermates and the frequency of lung metastasis was greatly reduced. When the mice were injected with the cells at 4 to 7 months, the primary tumor grew at the same rate in obese and lean mice, but the obese mice again showed a significantly reduced frequency of lung metastasis. That this effect was related to an enhanced immunocompetence in obese mice was supported by the finding that splenic lymphocytes of ob/ob mice showed three times the proliferative response to the T-cell mitogen concanavalin A compared with the proliferative response of lean control mice. The ob/ob mouse may provide a model for the study of enhanced immunocompetence in obese individuals.

Tumorigenicity of simian virus 40-hepatocyte cell lines: effect of in vitro and in vivo passage on expression of liver-specific genes and oncogenes.

Five simian virus 40 (SV40)-hepatocyte cell lines were examined for tumorigenicity and the effect of in vitro passage on the expression of four liver-specific genes (albumin, transferrin, alpha 1-antitrypsin, and phosphoenolpyruvate carboxykinase), two oncogenes (c-Ha-ras and c-raf), and two genes associated with hepatocarcinogenesis (alpha-fetoprotein and placental-type glutathione-S-transferase). At low passage (12 to 22), all five cell lines expressed the four liver-specific genes at levels similar to those in the liver and were not tumorigenic or were weakly tumorigenic. At high passage (33 to 61), the cell lines formed carcinomas, and four out of five cell lines produced primary tumors that metastasized. At least two cell lines produced well-differentiated hepatocellular carcinomas that expressed liver-specific RNAs. Levels of expression of liver-specific genes changed with time in culture. Some of the changes in liver-specific gene expression in the tumor tissue (such as for the phosphoenolpyruvate carboxykinase gene) paralleled those that occurred with in vitro passage, while other changes (such as for the albumin gene) did not parallel those that occurred with in vitro passage. Correlations between enhanced expression of c-Ha-ras and tumorigenic potential and between the process of SV40 immortalization and induced expression of c-raf and glutathione-S-transferase-P were observed. Induction of alpha-fetoprotein was detected with in vitro and in vivo passage only in the CWSV14 cell line and was paralleled by diminished albumin expression. In conclusion, we developed a model system with five SV40-hepatocyte cell lines, tumors induced by them, and tumor cell lines to examine changes in gene expression that accompany the progression from a normal cell to a hepatocellular carcinoma. Because the SV40-hepatocyte cell lines and tumor cell lines remain highly differentiated and vary in the magnitude of expression of specific genes, they can be used to study the molecular mechanisms regulating gene expression, in particular those regulating specific genes associated with differentiation.

Spontaneous maturation and differentiation of B16 melanoma cells in culture.

B16 melanoma tumors and cultures are composed of cells with different melanin contents and replicative activities. The hypothesis was tested in vitro that these various cells constituted a population in the process of differentiation and maturation. Early cultures were predominantly composed of small, amelanotic cells with high replicative activity. Older cultures contained mostly larger and heavily melanotic cells with little or no replicative activity. Replicative activity, as measured by the uptake of tritiated thymidine, was inversely proportional to cell size and melanin content. Colony-forming ability was impaired if the original cells were melanotic. Tumorigenicity was unaffected except in very old (9-day) cultures. Our results support the concept that malignant melanocytes undergo a sequence of developmental changes which eventuates in the production of mature cells characterized by enlargement, elevated melanin content, and reduced replicative and colony-forming capacity.

Increased immunogenicity of a spontaneous variant clone of the 13762A rat mammary adenocarcinoma.

The 13762A rat mammary adenocarcinoma is weakly immunogenic and spontaneously metastasizes to regional lymph nodes and lungs. A clone (18A) was isolated from the parental tumor, which grew for 3 weeks in normal F344 rats, forming tumors up to 2-3 cm and some nodal metastases, and then completely regressed. Pretreatment of recipient rats with 450 rad permitted progressive growth and death due to metastases. The behavior of 18A has been stable during a period of 120 days in continuous culture or for 6 in vivo passages in irradiated rats. Regression of 18A was associated with intense tumor mononuclear leukocytic infiltration, whereas parental tumors of the same size recruited few leukocytes. Regressions occurred when 18A cells were placed intradermally, sc, or im, but iv injections were not rejected. Parental tumors grew progressively at all sites. Regressor rats were specifically immune to challenge with both 18A and parental tumor but not to an unrelated mammary carcinoma (R3230AC). Irradiated 18A tumor cell vaccines protected recipients against challenge with parental tumor, but similar vaccines of irradiated parental tumor cells were ineffective. The systemic adoptive transfer of immune lymphocytes more strongly inhibited the growth of established (7 days) 18A than parental tumor. It was concluded that the parental 13762A tumor contained stable variants that were significantly more immunogenic and more susceptible to immune attack than the parental tumor. Such variant tumor lines may be useful in the study of the host response to metastasis.

Role of T-cell subsets in the destruction of established metastases of 13762A rat mammary adenocarcinoma.

The 13762A rat mammary adenocarcinoma metastasizes with high frequency to regional lymph nodes and lungs. The intratumoral injection of Corynebacterium parvum on day 7 followed by primary tumor excision on day 20 significantly prolonged survival and cured 10-40% of syngeneic F344 rats. Established metastases were destroyed by the treatment, and strong and specific tumor rejection immunity was induced. The purpose of the present study was to determine if T-cells were required for the C. parvum treatment to be effective and to identify the subsets of T-lymphocytes that might participate in the response. The results indicated that rats depleted by either neonatal thymectomy or a combination of adult thymectomy, 900 rad, and bone marrow reconstitution did not inhibit tumor growth after C. parvum treatment. Restoration of depleted rats with lymph node cells permitted effective treatment. The lymph node cells that were responsible for restoration expressed both W3/13 (pan-T-cell) and W3/25 (helper T-cell) membrane-associated differentiation antigens. T-cells that bore the MRC OX8 (cytotoxic-suppressor T-cell) antigen did not restore the response to C. parvum treatment. The effect of lymph node restoration was markedly potentiated by simultaneous administration of thymocytes, a T-cell population that expresses both W3/25 and MRC OX8 antigens. In conclusion, the cytotoxic-suppressor T-cells were ineffective in the restoration of T-cell-depleted, tumor-bearing rats to benefit from C. parvum but helper T-cells were highly effective, and their activity was strongly potentiated by administration of thymocyte amplifier cells.

Evaluation of antitumor activity of Bordetella pertussis in two murine tumor models.

The antitumor activity of three preparations of killed Bordetella pertussis (Bp) (Eli Lilly crude and fluid pertussis vaccines and Parke-Davis pertussis vaccine) was studied in the B16 melanoma and CaD2 mammary adenocarcinoma models. In these tumor systems; Bp had weak and variable tumor inhibitory activity and did not augment tumor rejection immunity. The intratumor injection of Bp did not affect the growth of the B16 tumor but significantly inhibited the growth of the CaD2 tumor. However, the established tumor did not regress. Admixture of Bp with B16 cells before inoculation inhibited tumor growth and prolonged survival of inoculated mice. Admixture of Bp with CaD2 cells completely suppressed tumor cell growth in 60% of inoculated mice. Intratumor injection of CaD2 with Bp combined with surgery provided no protection against subsequent development of metastases.

Runt syndrome induced in the rat by polyinosinic-polycytidylic acid.

Polyinosinic with polycytidylic acid (poly l with poly C), a double-stranded synthetic RNA, produced in newborn rats a runt syndrome characterized by mortality and retarded growth rates of the total body, thymus, and kidneys. In contrast, it induced a hyperplasia in the epidermis and in the spleen. Within 10 days of treatment, the epidermis became 2 or 3 times thicker and the spleen mass was increased by 50%. The epidermal hyperplasia involved all layers, but hair follicles were excluded. Splenic hyperplasia did not result from accelerated erythropoiesis. Double-stranded RNA was required; single-stranded homopolymers were ineffective. Theophylline, a phosphodiesterase inhibitor, did not potentiate the effects. The uptake of iododeoxyuridine-125 was not enhanced in the hyperplastic epidermis or spleen. Thus we concluded that poly l with poly C can retard the growth of some organs in newborn rats, but that it causes epidermis and spleen to accumulate cells. The cytokinetic mechanisms involved in these contrasting effects were not clear.

Inconsistent response of B16 melanoma to BCG immunotherapy.

We evaluated the potential of the B16 melanoma of mice as a model system for BCG immunotherapy of malignant melanoma. We studied a variety of treatment protocols: a) BCG given simultaneously but separately with a small number of B16 cells significantly inhibited tumor growth in only three of eight experiments. b) BCG injected directly into the tumor stimulated tumor growth in three of three experiments; the stimulation was at least partially attributable to the nutrient medium in which the BCG was suspended. c) The B16 tumor was weakly immunogenic and the addition of BCG to a tumor cell vaccine offered little improvement in subsequent resistance to tumor cell challenge: d) In a model of postsurgical residual tumor, metastatic to regional lymph nodes, BCG and tumor cell vaccination did not alter the development of nodal metastases. The B16 melanoma was not a useful model system for BCG immunotherapy, because the tumor inhibition was feeble, inconsistent, and not associated with augmented tumor immunity.

Suitability of rat mammary adenocarcinoma 13762 as a model for BCG immunotherapy.

We determined the optimal conditions for conducting experiments with the solid and ascites sublines of the 13762 rat mammary adenocarcinoma and examined the response of the tumor growth rate to BCG administered in admixture with tumor cells or separately at a remote site. Versene dissociation of the 13762 solid tumor produced better growth rates than did pronase-DNase, but the former decreased cell viability and yields. A dose of 10(6) or 10(5) tumor cells produced 100% growth by the sc and iv routes. Both sublines grew slower but produced metastases slightly sooner in the intradermal than in the sc site. The frequency of axillary lymph node metastases from the sc site increased as a function of the duration of the time interval between tumor implantation and surgical excision. Both solid and ascites tumors were weakly immunogenic. Administration of BCG in a split adjuvant protocol did not improve tumor immunity. Admixture of tumor cells with BCG suppressed tumor growth but when given at a remote site, BCG was ineffective. We concluded that the 13762 rat mammary adenocarcinoma is a useful system for BCG immunotherapy.

Relationship between intradermal tumor suppression and tumor immunity.

Intradermal (id) injection of three tumor-immune stimulant mixtures (LSTRA-BCG, 13762A-BCG, CaD2-Corynebacterium parvum) was superior to the sc site for suppression of tumor growth: Suppression of LSTRA-BCG mixtures was even less efficient after an ip or iv injectiouppression at all four sites. In the LSTRA-BCG model, the id site was not uniquely favorable for either the afferent or efferent limb of the immune response; the other sites produced equally effectiveimmunization or rejection of tumor challenge. We concluded that local suppression of tumor cell-immune stimulant mixtures was frequently more effective in the skin than at other sites, that local tumor suppression did not depend primarily on tumor immunity, and that afferent and efferent tumor immunity were equally efficient by the four routes tested.

Maturation and differentiation of B16 melanoma cells induced by theophylline treatment.

Recent studies suggested that 3',5'-cyclic AMP (CAMP) may be involved in the regulation of cell proliferation and differentiation. Theophylline, an inhibitor of cyclic nucleotide phosphodiesterase, elevated intracellular cAMP. A melanotic clone of the B16 melanoma was treated with theophylline and studied in vitro and in vivo. With 12 hours after 1.0 mM theophylline was added to growing cultures, the number of cells incorporating tritiated thymidine (3-H-TDR) and the rate of uptake of 3-H-TDR into DNA were significantly reduced. After 7 days, the number of cells in the control cultures increased twenty-four times, whereas theophylline-treated cells increased only sixfold. Compared to the controls, the theophylline-treated cells contained ten times the melanin and an elevated cAMP content. Stimulation of melanogenesis and inhibition of proliferation increased progressively with duration of exposure to theophylline. After 5 days of culture with theophylline, cells were assayed for plating efficiency in theophylline-free medium. Although the number of colony-forming cells was unaffected by previous exposure to theophylline, the colonies were composed of fewer cells inoculated into syngeneic hosts were less tumorigenic than untreated cells. However, theophylline treatment of hosts bearing B16 tumors failed to reduce the tumor growth rate, and theophylline did not potentiate the growth inhibition resulting from treatment with the synthetic polyribonucleotide, polyinosinic-polycytidylic acid.

Detection of virus-specific RNA in herpes simplex virus type 1-transformed hamster cell lines.

In situ hybridization experiments with the use of recombinant herpes simplex virus type 1 DNA as probes have detected virus-specific RNA in herpes simplex virus type 1-transformed Syrian hamster cell lines. The relatively most abundant virus transcripts hybridized to the herpes simplex virus type 1 EcoRI-F fragment at map position 0.32-0.42.

Antitumor activity of killed Corynebacterium parvum suspensions in a murine mammary adenocarcinoma CaD2) system.

We studied the antitumor activity of killed Corynebacterium parvum on the CaD2 mammary adenocarcinoma in DBA/2 mice. Intratumor treatment had little or no effect on subcutaneous tumor growth. Admixture of tumor cells with C. parvum before inoculation completely suppressed tumor growth. No tumor transplantation immunity was detected in mice inoculated with admixtures of C. parvum and tumor cells, but tumors were enhanced under certain circumstances. Growth of a tumor inoculated sc or iv was consistently retarded after iv or ip C. Parvum therapy, but tumors rarely regressed. Tumor transplantation immunity was detected in mice treated iv with C. parvum before surgical excision of established tumors. Certain adverse effects of iv or ip administration of C. parvum were also discussed.

Properties of human epithelioid cells established in vitro by a herpesvirus [IBRV(HMC)] isolated from cytomegalovirus-transformed human cells.

Infectious bovine rhinotracheitis virus [IBRV(HMC)], a double-enveloped herpesvirus, was isolated from human embryo lung fibroblasts transformed by cytomegalovirus (CMV). This agent was identified as an IBRV strain that was antigenically related to human CMV. Inoculation of a primary human kidney cancer cell culture with IBRV(HMC) resulted in persistent infection and subsequent establishment of a cell line [IBRV(HMC)HKC-1]. Virus-related nuclear, cytoplasmic, and cell membrane antigens were detected in these cells in early in vitro passages by anticomplement and indirect immunofluorescence tests. Infectious virus was rescued from one of the cell sublines after temperature-shock treatment at passage 26. Karyotypic analysis confirmed the human origin of the cells. Control uninfected kidney cancer cells survived only six in vitro passages. The established cells grew to more than 100 in vitro passages 1 year after initiation of the experiments and induced an epithelioid cancer of variable morphology that infiltrated nerves and muscles when inoculated sc into athymic nude mice.

Systemic adoptive transfer of immunity to 13762A rat mammary adenocarcinoma.

Rats cured of metastatic 13762A rat mammary adenocarcinoma by Corynebacterium parvum immunotherapy possess strong tumor-specific rejection immunity. Systemic adoptive transfer of lymphoreticular cells from cured rat donors conferred protective immunity on naive recipients. Fewer oil-induced peritoneal exudate cells than lymph node cells were required to transfer tumor rejection immunity. The adoptive immunity was specific since it strongly inhibited 13762A tumor growth but did not inhibit the growth of the antigenically unrelated R3230AC rat mammary tumor. Rats sensitized to the bacterial immune stimulant used to effect cure were unsuitable donors of PEC capable of transferring tumor rejection immunity. Macrophages or bone marrow-derived lymphocytes from immune donors did not transfer immunity. Treatment of peritoneal exudate with a xenogeneic antiserum specific for rat thymus-derived lymphocytes significantly reduced the efficiency of transfer. We conclude that thymus-derived lymphocytes from rats cured of 13762A tumor were required for the adoptive transfer of tumor-specific rejection immunity.

Regulation of the expression of adoptive tumor rejection immunity by recipient cyclophosphamide-sensitive cells.

Peritoneal exudate T-cells from rats immune to 13762A rat mammary tumor conferred specific tumor rejection immunity on normal recipients. The efficiency of systemic adoptive transfer of tumor rejection immunity with immune peritoneal exudate T-cells was improved by cyclophosphamide (CY) pretreatment of recipients. Optimal potentiation was obtained with a dose of 50 or 100 mg CY per kg body weight given the day before transfer. CY pretreatment of recipients was effective 1 to 3 days prior to transfer. The CY-potentiating effect was lost with longer intervals between CY administration and transfer, indicating recipient recovery. CY pretreatment enabled recipients to reject greater numbers (100 times) of tumor cells and inhibited tumor challenge established before systemic adoptive transfer. The CY-induced potentiation of systemic transfer of tumor immunity was reversed by i.v.-administered normal spleen. We concluded that CY-sensitive host regulatory cells restricted the expression of adoptive tumor rejection immunity. Control of the activity of these regulatory cells allows increased efficacy of effector T-lymphocytes in this system.

Effects of dose and schedule of immune stimulant on efficacy of combination Corynebacterium parvum-cyclophosphamide treatment for a murine mammary adenocarcinoma.

Certain variables which might influence the outcome of combining cytotoxic drug and immune stimulant therapy were studied to optimize the effectiveness of Corynebacterium parvum combined with cyclophosphamide (CY) as treatment for a murine mammary adenocarcinoma (CaD2). Optimal effects of combined C. parvum-CY treatment in the CaD2 system were obtained when 443 to 1400 microgram of this immune stimulant per mouse were injected 2 to 3 days after CY chemotherapy and when combination treatment was continued on a weekly basis. The most critical factors contributing to the effectiveness of combination treatment in this system were the dose of C. parvum and the treatment frequency. The interval between chemotherapy and immune stimulant therapy was less critical to the outcome of combination treatment. Combination treatment given once or weekly significantly decreased tumor size in comparison to single or weekly CY treatment. A single treatment with CY and C. parvum significantly improved the survival over mice given a single CY treatment, but weekly CY and C. parvum treatment did not increase the survival over mice, given weekly chemotherapy.

Tumorigenicity of simian virus 40-transformed rat hepatocytes.

Simian virus 40 (SV40)-transformed F344 rat hepatocytes were transplanted into newborn and normal and irradiated adult syngeneic F344 hosts. Three independently isolated SV40-transformed hepatocyte cell lines were inoculated s.c. into newborn syngeneic animals. One cell line, SV40hpl, produced tumors in two of 12 animals. Fixed tissue sections indicated that the tumors were relatively undifferentiated but had some epithelial cells and contained a peripheral mononuclear leukocyte infiltrate. Serum from a tumor-bearing animal tested by immunoprecipitation demonstrated antibodies to SV40 large-T- but not to small-t-antigen. A portion of one of the tumors was used to prepare the tumor cell line SV40hpl-1, which was 100% positive by immunofluorescence for SV40 nuclear T-antigen. In adult F344 rats subjected previously to whole-body irradiation, inoculation of SV40hpl-1 cells produced tumors in 83% of the animals. Continued passage of the tumor cell line in irradiated rats produced a tumor in one animal which paralyzed its host. The cell line derived from this tumor (SV40hpl-1-T1-2) was tumorigenic in nonirradiated adult hosts. The tumor cell lines were morphologically similar to each other but different from the SV40hpl-transformed cell line; the tumor cells contained less cytoplasm and grew to high densities in strings or multilayered foci without covering the dish surface. All tumor cells retained SV40 antigen expression, and at least one complete copy of the virus genome was retained through two passages in animals. We concluded that SV40 transformation of rat hepatocytes can produce a tumorigenic cell line. We have shown previously that SV40-transformed hepatocytes retain the ability to express proteins characteristic of adult differentiated liver. Development of this model system will enable us to examine expression of these proteins not only in transformed cells with tumorigenic potential but also in tumor tissue and transplantable tumor cell lines.

Primary neoplastic transformation in vivo of xenogeneic skin grafts on nude mice.

Rabbit skin was infected with Shope papilloma virus and grafted orthotopically to nude mice. Typical Shope rabbit papillomas developed in most of the grafts, but similarly treated nude mouse skin grafts were not altered. These results demonstrated that xenogeneic tissues retained susceptibility to oncogenic agents after transplantation to nude mice. The results also implied that it should be feasible to study carcinogenesis of human tissues in this system.