The problem of enhancing the biological action of ionizing radiations. Glucose as an agent for increasing the radiosensitivity of Ehrlich ascitic carcinoma cells in vitro

The effect of short-term incubation of Ehrlich ascites tumor cells in a medium containing excess glucose on their radiosensitivity was studied with a reference to the growth of tumors of ascitic and solid forms. It was shown that the incubation of cells with glucose, being accompanied by a change in pH of the suspension, caused, by itself, only a slight increase in the duration of the latent period of solid tumor formation and in the life-span of mice with ascitic tumors but increased considerably the lethal effect of radiation, as was estimated by the above mentioned criteria and the percentage of inoculated tumors.     

一些抗癌药物对荷瘤小鼠血清中唾液酸含量的影响

测定荷六种小鼠肿瘤S180肉瘤(实体型和腹水型),腹水肝癌(HepA),艾氏腹水瘤(EC),白血病P388和Lewis肺癌的小鼠腹水和血清中唾液酸含量,结果显示血清中唾液酸含量与肿瘤生长、肿瘤类型有关。腹水中唾液酸含量高,推测肿瘤能比正常组织产生更多唾液酸。对四种腹水肿瘤用阴离子交换树脂层析鉴定,发现HepA腹水中葡萄糖代唾液酸(NcuGc)含量明显低于其它三种腹水瘤。还研究了十几种抗癌药物对荷S180和Lewis肺癌小鼠血清中唾液酸含量的影响。发现吗丙嗪(probimane)和顺铂(DDP)能降低荷瘤小鼠血清中唾液酸含量,提示此二药物在肿瘤治疗中更具选择性。     

Cytotoxic T lymphocytes in DBA/2 mice harboring L5178Y cells in a tumor-dormant state

Previous experiments have demonstrated a temporal relationship between the decline of cytotoxic T lymphocyte (CTL) activity in the peritoneal cavity of DBA/2 mice harboring L5178Y cells in a tumor-dormant state and the appearance of ascitic tumors. Some tumor-dormant mice remain clinically normal for many weeks after the decline of CTL activity, and this activity can be rapidly restimulated by an IP inoculation of irradiated L5178Y cells. We report here that the peritoneal cells from many tumor-dormant mice can be stimulated to cytolytic activity in vitro when cultured for 4 days either with or without the addition of irradiated L5178Y cells. Peritoneal cell populations which cannot be stimulated in vitro can suppress the generation of CTL in those populations which can be stimulated. The tumor-dormant state may terminate when suppressor cells in the peritoneal cavity of tumor-dormant mice inhibit the generation of CTL activity and permit tumor cells to produce an ascitic tumor.     

Differential expression of fibronectin in a rat histiocytoma: possible role of fibronectin in tumor cell adhesion

The specific role of fibronectin in tumor cells has been investigated using the transplantable histiocytic tumor line AK-5 [A. Khar (1986) J. Natl. Cancer Inst. 76, 871]. These cells, capable of growth as both ascites and solid tumors in rats, can be separated into four subpopulations by buoyant density centrifugation on Percoll. These subpopulations are shown to contain different amounts of fibronectin on the cell surface when studied by immunofluorescent staining followed by cytofluorometric analysis. Cells which contain high amounts of fibronectin can grow both as ascites and as solid tumors while those cells which contain low amounts of fibronectin cannot grow as solid tumors but can grow as ascitic tumors. Pretreatment of high-fibronectin-containing cells with anti-fibronectin antibody abolished their capacity to grow as solid tumors; however, the cells retained their capacity to grow as ascitic tumors. These results have been discussed with reference to the specific role of fibronectin in tumor-forming cells.     

Cytotoxic T lymphocytes in DBA/2 mice harboring L5178Y cells in a tumor-dormant state

Summary Previous experiments have demonstrated a temporal relationship between the decline of cytotoxic T lymphocyte (CTL) activity in the peritoneal cavity of DBA/2 mice harboring L5178Y cells in a tumor-dormant state and the appearance of ascitic tumors. Some tumor-dormant mice remain clinically normal for many weeks after the decline of CTL activity, and this activity can be rapidly restimulated by an IP inoculation of irradiated L5178Y cells. We report here that the peritoneal cells from many tumor-dormant mice can be stimulated to cytolytic activity in vitro when cultured for 4 days either with or without the addition of irradiated L5178Y cells. Peritoneal cell populations which cannot be stimulated in vitro can suppress the generation of CTL in those populations which can be stimulated. The tumor-dormant state may terminate when suppressor cells in the peritoneal cavity of tumor-dormant mice inhibit the generation of CTL activity and permit tumor cells to produce an ascitic tumor. Abbreviations used in this paper: C, complement; CTL, cytotoxic thymus-derived lymphocyte; PC, peritoneal cells; DPC, days post challange; NAD, nonadherent; SC, subcutaneous; IP, intraperitoneal     

Intracamerally induced concomitant immunity: mice harboring progressively growing intraocular tumors are immune to spontaneous metastases and secondary tumor challenge

Intracameral inoculation of allogeneic P815 mastocytoma cells (DBA/2) into BALB/c mice resulted in progressively growing intraocular tumors. Intraocular tumor cells disseminated rapidly to the spleen and cervical lymph nodes, yet extraocular nests of tumor cells never developed into fulminant tumors. Further experiments showed that tumor cells were continuously seeded from the primary intraocular tumor and were rapidly cleared from extraocular sites. Hosts harboring intraocular P815 mastocytomas rejected tumorigenic doses of P815 cells inoculated subcutaneously or even into the contralateral anterior chamber. This systemic tumor immunity was found to be radiosensitive and T cell dependent. Spleen cells from animals with progressively growing intraocular tumors protected recipient mice challenged with intracamerally inoculated tumor cells and thus suggests that a cell-mediated mechanism is the underlying basis for this form of tumor immunity. The data indicate that mice harboring progressively growing intraocular tumors develop a potent state of "concomitant immunity," that prevents the development of metastases, yet is ineffective in controlling the primary tumor.     

Cytogenetic analysis of an immunogenic mutant of the L5178Y lymphoma

A mutant of the uniformly lethal L5178Y lymphoma, called the L5178Y/Manitoba (L5178Y/M), was rejected after subcutaneous challenge in syngeneic DBA/2 mice. Karyotypic analysis revealed that the parent L5178Y lymphoma had four chromosome markers, with the mutant L5178Y/M sharing one of them as well as possessing two distinguishing markers. One diploid and two hypotetraploid clones were isolated from the L5178Y/M; they contained all the marker chromosomes and were also rejected by the syngeneic host. In addition to the shared chromosome markers, the L5178Y/M possessed antigens in common with the parent L5278Y. DBA/2 mice made immune to the mutant by subcutaneous immunization were able to slow the growth of the parent tumor but not the unrelated P-815-X2 mastocytoma.     

Comparative studies of mouse tumors with respect to their capacity for growth as “ascites tumors” and their average nucleic acid content per cell

The advantages presented by the use of the comparatively homogeneous free tumor cell suspension of the Ehrlich ascites tumor for quantitative studies on the growth and chemical composition of tumor cells and for chemotherapeutic experiments have led to the present investigation of a representative array of 26 histologically and genetically different mouse tumors which were tested for their capacity of reaching a condition similar to the Ehrlich ascites tumor. This has been found to be possible in a number of cases. Dissociated solid tumor masses were intraperitoneally inoculated into the corresponding mouse strain. Tumor growth on the peritoneum, accompanied by exudate formation, resulted in 22 out of 26 cases. The resulting exudates, which, in the case of all carcinomas and most sarcomas tested, contained very few tumor cells, transmitted the tumors upon further intraperitoneal injection in 18 instances. The tumors which developed were now regularly provoking exudate formation, and on subsequent generations the exudates have always been transplanted intraperitoneally. In the case of all carcinomas and in 2 out of 3 sarcomas tested which have passed over 10 such “fluid transplant generations” (FTG), a gradual increase in the frequency of tumor cells in the ascitic fluids (examined around median survival time) was occurring after a certain     

The negative systemic effect of BCGcw inoculated intraperitoneally

Summary The present studies describe a systemic effect of BCGcw inoculated intraperitoneally by observing its influence on the development of Morris hepatomas inoculated intramuscularly. In none of our studies did we observe an inhibition of tumor growth. Instead, an enhancement of tumor growth was seen with four antigenically distinct Morris hepatomas (3924a, 9098, 7777, and 5123tc) in two strains of inbred rats (Buffalo and ACI). Extensive studies with Morris hepatoma 3924a showed consistent enhancement of intramuscular tumor growth in eight of eight experiments with a tumor cell dose of 1×10⁵ and a BCGcw dose of 900 g. In BCGcw-inoculated animals, palpable tumors (1–2 g) were detected approximately 1 week earlier than in controls, and significantly larger tumor masses were noted on the day of sacrifice. With the threshold dose of cells, 1×10⁴, an increase in tumor incidence was observed. It was not necessary for the host to be immunized with BCGcw prior to tumor inoculation as enhancement of tumor growth occurred when the BCGcw were inoculated the same day or 7 days after tumor inoculation. Splenectomized animals also demonstrated BCGcw-mediated enhancement of tumor growth. BCGcw immunization blocks the induction of tumor-specific immunity. When 3924a ascites tumor cells were inoculated intradermally into normal animals, no tumor growth was observed and tumor-specific immunity resulted. When 3924a ascites tumor cells were inoculated intradermally into BCGcw-immunized animals, progressive intradermal tumors grew in all the animals, implying that tumor-specific immunity was not induced. The administration of BCGcw did not effect established tumor-specific immunity to line 3924a as assessed by tumor-specific rechallenge resistance. Studies with an allograft system showed that the ACI tumor 3924a would not grow in normal Buffalo rats, but transient tumor growth was observed when the Buffalo rats were immunized with BCGcw. The abbreviations used are: BCGcw, bacillus Calmette-Guérin cell walls attached to oil droplets; MLC, mixed lymphocyte culture; MER, methanol extract residue; IM, intramuscular; IP, intraperitoneal; ID, intradermal; Con-A, concanavalin-A     

Solid intraperitoneal Landschütz tumour in mice: implications in cancer chemotherapy and immunology studies.

Multiple intraperitoneal injections of various normal sera into BALB/c mice inoculated intraperitoneally with Landschütz ascites tumour cells abrogated the development of ascitic syndrome in almost all the animals. In a large proportion of the survivors solid intraperitoneal tumours developed, composed of characteristic ascites tumour cells engulfed and encapsulated in connective tissue. The effect of serum on the development of the solid tumour was diminished if the donor had been immunized against mouse IgG. Inoculated animals treated with serum hyperimmune against mouse IgG showed accelerated ascitic tumour growth. Cyclophosphamide or arabinosylcytosine strongly inhibited growth of solid tumours. Simultaneous administration of arabinosylcytosine and its antagonist cycloheximide did not interrupt tumour growth.     

Characterization of L5178Y leukemic cells which rapidly develop and lose implantation ability.

Two populations of L5178Y murine leukemic cells, maintained by different methods, were studied for their implantation ability in BDF₁ mice. Implantation ability was measured by number of tumor nodules formed, liver weight, and day of death of the animal. 1) Cells from a population grown for 10 years $\text{in vitro}$ had no implantation ability; i.e., no tumor nodules were formed when injected into the tail vein. After 30 days of growth in the peritoneal cavity of BDF₁ mice, these same cells were injected into the tail vein and 10 days later had produced over 200 liver tumor nodules. When cells taken from these tumors were recultured for 60 days $\text{in vitro}$, they lost the acquired implantation ability, but regained it after another single peritoneal passage. 2) L5178Y murine leukemic cells grown for six years in ascites tumor cells were extremely tumorigenic; over 200 tumor nodules appeared in the liver after tail vein injection. These cells were not rendered less tumorigenic and did not lose their implantation ability by $\text{in vitro}$ culturing for 60 days. The results suggest that implantation ability is a property of the cell's growth environment; furthermore, they have strong implications for the $\text{in vivo}$ and $\text{in vitro}$ manipulation of this property.     

Differential anti-proliferative properties of novel hydroxydicarboxylatoplatinum(II) complexes with high or low reactivity with thiols

We have examined the anti-proliferative effect of 13 recently synthesised platinum dicarboxylate complexes, very similar in their chemical, structural and kinetic properties to carboplatin. We used the L5178Y model: two murine lymphoma sublines, which differ in nucleotide excision repair ability and hence, in sensitivity to those platinum complexes that react with DNA. The anti-proliferative effect of the examined compounds mainly depends on the kind of amine ligand. Complexes with the primary amine (ethylenediamine) are more effective than complexes containing the tertiary amine (1-alkylimidazole). The ethylenediaminemalatoplatinum(II) complexes show a differential in vitro anti-proliferative activity in the L5178Y model; hence, it may be expected that they inflict DNA lesions that are repaired by the nucleotide excision system. The cytotoxicity of these complexes is directly correlated with reactivity with glutathione (GSH). The 1-alkylimidazole complexes are of low toxicity and moderate to low reactivity with GSH; in contrast to the ethylenediaminemalatoplatinum(II) complexes, their cytotoxicity is inversely correlated with reactivity with GSH. Two of the 1-alkylimidazole complexes, bis(1-ethylimidazole)(L-malato)platinum(II) and bis(1-propylimidazole (L-malato)platinum(II), show a considerable ability to arrest cells in G2 phase. We expect that the properties of these two groups of platinum complexes may be exploited in combined platinum complex treatment and irradiation.     

The modelling of the growth of Ehrlich carcinoma and teratoma T-36 with ascitic fluid and tumor-cell dialysate]

The study of the effect of ascitic fluid and dialysate of Ehrlich ascites tumor cells (M.m. less than 15 kDa) on the growth of Ehrlich carcinoma and teratoma T-36 has shown that both the ascitic fluid and dialysate can protect tumor cells in vivo. The number of animals with tumors increased from 0% in control animals to 60 and 20%, respectively, in experimental ones after transplantation i.m. of 20 x 10(3) Ehrlich tumor cells into mice. Compared to control, ascitic fluid and dialysate of Ehrlich ascites tumor cells increased the rate of tumor growth to 195 and 153%, respectively. It is suggested that this test-system simulates the effect of tumor humoral factors in vivo.     

Inactivation of glutathione reductase by 2-chloroethyl nitrosourea-derived isocyanates.

The specific inactivation of yeast glutathione reductase (GSSG-reductase) by 2-chloroethyl isocyanate and cyclohexyl isocyanate derived from their respective 2-chloroethyl nitrosoureas has been demonstrated. Titration of the enzyme with 2-chloroethyl isocyanate or [¹⁴C] labeling with 1-(2-chloroethyl)-3-(1-¹⁴C-cyclohexyl)-1-nitrosourea or 1,3-bis (2-¹⁴C-chloroethyl)-1-nitrosourea resulted in near stoichiometric inactivation and/or covalent labeling of the enzyme. In addition to 1,3-bis (2-chloroethyl)-1-nitrosourea and 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea, several other 2-chloroethyl nitrosoureas were capable of inactivation of not only purified GSSG-reductase, but also the activity of this enzyme in cell-free extracts of murine lymphoma L5178Y ascites tumor cells and murine bone marrow.     

Conformational changes in the DNA of hybridoma cells from pristane treated mice

The effects of pristane on the DNA of hybridoma cells propagated as ascitic tumors in pristane-primed BALB/c mice were determined using flow cytometric analyses. Hybridoma cells maintained in vitro or cell isolates from solid tumors which developed in unprimed mice injected with hybridoma cells exhibited similar propidium iodide (PI) staining characteristics. In contrast, PI stained cells isolated from ascites which developed in pristane-primed mice injected with the hybridoma cells displayed significant decreases in fluorescence intensity. Diphenylamine studies and analyses of pH 10 treated cells indicated that the actual DNA content of the hybridoma cells was not altered by exposure to pristane. Furthermore, the altered staining characteristics of the ascitic tumor cells were reversible in that the fluorescence intensity after serial in vitro passage of the ascites cells was similar to that of the parent cell line which had not been exposed to pristane. In addition, there was a direct correlation between the altered PI staining characteristics and the presence of cell-associated pristane as determined by gas-liquid chromatography analyses of cell extracts. Collectively these results suggest that pristane may have a direct effect on the DNA conformation of hybridoma cells which may in turn enhance their growth as ascitic tumors. The possible role of such an altered DNA conformation in hybridoma cells on the in vivo development of ascites is discussed.     

Comparative radiosensitivity of clonogenic tumor cells of the transplantable Ehrlich carcinoma in the ascitic and solid forms of growth

Survival of clonogenic cells of solid Ehrlich ascites tumor (EAT) exposed to 60Co-gamma-radiation in vitro under the oxygenation conditions was investigated and the clonogenic capacity and radiosensitivity of these cells and cells of the previously studied EAT ascitic form and Lewis solid tumor comparatively studied to elucidate how the efficiency of colony formation (ECF) would affect their radiosensitivity. ECF for solid EAT cells was 2.6 +/- 0.3%, which was lower, by about an order of magnitude, than that for ascitic form of this tumor and was nearly the same as that for Lewis tumor cells. A median cell lethal dose (D0) was practically the same for all tumors under study. It is suggested that the differences in ECF do not substantially influence the radiosensitivity of clonogenic cells of the studied tumors.     

Analysis of protective and cytotoxic immune responses in vivo against metabolically inactivated and untreated cells of a mutagenized tumor line (requirements for tumor immunogenicity)

The immunogenicity of a mutagenized subline (ESb-D) of the weakly immunogenic T-cell lymphoma L 5178 Y ESb has been characterized. The injection of 10(6) ESb-D cells ip did not establish lethal tumors in untreated DBA/2 mice but established tumors in sublethally irradiated mice. Injection of ESb-D cells into otherwise untreated DBA/2 mice established also a state of protective immunity against the subsequent injection of otherwise lethal doses of ESb tumor cells. Protection was only obtained after injection of intact but not UV-irradiated or mitomycin-C-treated ESb-D cells. A direct T-cell-mediated cytotoxic activity was also demonstrable in the spleen cells of DBA/2 mice after injection of ESb-D cells but not ESb cells. The cytotoxic activity was variant specific for ESb-D target cells, and it was induced only with intact but not UV-irradiated or mitomycin C-treated ESb-D cells. This suggested that the induction of protective and cytotoxic immunity may require the persistence of the antigen or unusually high antigen doses. The in vivo priming for a secondary in vitro cytotoxic response, in contrast, was achieved with intact and also with mitomycin C-treated ESb-D cells but again not with UV-irradiated ESb-D cells. This indicated that the metabolic activity was a minimal requirement for the in vivo immunogenicity of the ESb-D tumor line. The secondary cytotoxic activity was demonstrable on ESb-D and ESb target cells and could be restimulated in vitro about equally well with ESb-D and ESb cells. But the in vivo priming was again only obtained with ESb-D cells and not with ESb cells. These experiments thus demonstrated that the requirements for immunogenicity are more stringent in vivo than in vitro, and more stringent for the induction of direct cytotoxic and protective immunity in vivo than for the in vivo priming for secondary in vitro responses.     

Immune regulation of the L5178Y murine tumor-dormant state. II. Interferon-gamma requires tumor necrosis factor to restrain tumor cell growth in peritoneal cell cultures from tumor-dormant mice

L5178Y lymphoma cells are restrained from progressive growth in peritoneal cell ("in vitro tumor-regressor" PC) cultures prepared from many DBA/2 mice which harbor the tumor cells in the peritoneal cavity in a tumor-dormant state. Treatment of these PC cultures with 'antibodies to murine interferon-gamma (MuIFN-gamma) and murine tumor necrosis factor (MuTNF) but not with antibody to interleukin 2 (IL-2) receptors eliminated the restraint on tumor cell growth and permitted their progressive proliferation. L5178Y cells were found to be resistant to the direct toxic effects of large concentrations (3,000 U/ml) of MuIFN-gamma and of MuTNF, either alone or in combination. Treatment of PC cultures from tumor-dormant mice, in which tumor cells grew progressively ("in vitro tumor-progressor"), but not PC cultures from normal mice, with exogenous MuIFN-gamma resulted in a marked inhibition of tumor cell growth. The MuIFN-gamma-induced cytotoxic activity was cell-mediated since no soluble tumor-cytotoxic factors could be detected in the cultures. MuIFN-gamma induced cytotoxic activity in plastic-adherent peritoneal cell (AD-PC) cultures, but induced no cytotoxic activity in nonadherent-PC cultures unless small numbers (2%) of AD-PC were present, and inclusion of antibody to MuTNF in these mixed PC cultures blocked the development of cytotoxic activity. Antibody to MuTNF also blocked the development of cytotoxic activity in cultures of MuIFN-gamma-treated whole PC and AD-PC from tumor-dormant mice. These results indicate that MuIFN-gamma and MuTNF are both important in restraining tumor cell growth in PC cultures from tumor-dormant mice, and that MuIFN-gamma requires the presence of MuTNF to induce cytotoxic activity in these cultures.     

Cellular growth control: properties of a unique adhering derivative of L5178Y cells

A new cell line derived from L5178Y murine lymphoma cells with unique properties is described. The cells, L5178Y Adh, grow simultaneously in suspension culture and attached to a substratum but not in contact with each other. Limitation of growth of the cells in either the suspended state or attached to substratum is not linked to a limiting surface or cell density in a manner analogous to “contact inhibition.” The adhering and non-adhering cells grow with different doubling times. Cell size analysis indicates that initial gravity settling of the inoculum determines which cells attach. Contact of these cells' external surface with substratum may then initiate events which ensure attachment of adhering populations.     

Structure-activity relationship of polyamine derivatives of 1,3-dichloroacetone-thiosemicarbazone: induction of metastases and increase in sialylation of murine lymphoma L5178Y-R cells

A number of polyamine (PA) derivatives of thiosemicarbazone of 1,3-dichloroacetone (TDA) have been prepared and their effect on growth in vivo of tumorigenic but not metastatic cell strain (LY-R) of mouse lymphoma L5178Y has been investigated. Polyamine derivatives of TDA (PDT) were injected i.p. every third day (4 times, 10 or 25 mg/kg per injection) into DBA/2 mice inoculated i.p. or s.c. with LY-R cells. It has been found that disubstituted putrescine (Put), spermidine (Spd) and spermine (Spm) derivatives TDA exhibit a prometastatic activity as indicated by the appearance of solid tumor foci in subcutaneous tissues, liver and spleen. This activity depends mainly on the structure of the PA fragment and the presence of TDA. An increase in lipid bound sialic acid content after treating LY-R cells in vitro and in vivo with a Spm derivative has been found. These findings suggest that disubstituted PA derivatives of TDA and LY-R cells may be a useful model for investigation of the final steps in formation of metastases by lymphoma cells.