The effect of exogenous alkalization on cytokinetics and on the survival rate of mice with lymphoblastic leukemia]

In mice vaccinated with two forms of lymphoblastic leukaemia and alkalized with intravenous administration of sodium bicarbonate, the survival rate, the extent of leukaemic infiltration and the proliferative capacity of cells in the bone-marrow, thymus, spleen, lymphnodes, liver and lungs were investigated. The survival rate in the TAL leukaemia of the AKR stem producing an endogenous acidosis could be significantly prolonged in a statistical way by alkalization. Yet an accelerated expiring rate could be observed after exogenous alkalization in L-1210 leukaemia of the DBA/2J stem producing an endogenous alkalosis. By means of cytological and impulse-cytophotometrical investigations the exogenous alkalization of both forms of leukaemia could be proved to have a direct bearing on the proliferative kinetics. In TAL leukaemia the leukaemic proliferation was inhibited by the exogenously involved correction of the acid-base balance; in the L-1210 leukaemia, however, the pH disturbances were enhanced, thus accelerating the leukaemic proliferation. Consequently, the disturbances of the acid base balance seem to be an essential cofactor in the leukaemia genesis. The exogenous direction of the acid-base balance may be important as a means of treating leukaemia.     

Comparative characteristics of the antitumor and immunodepressive activity of carminomycin on the L-1210 experimental model]

The antitumor activity of carminomycin was estimated by the number of lymphoma colonies formed in the spleen of DBA/2 mice on their inoculation with the bone marrow cells from mice with transplantable leukemia L-1210. The immunodepressive properties of carminomycin were determined by the number of the antibody forming cells in the spleen of CBA and DBA/2 mice with leukemia L-1210 after immunization with sheep red blood cells. It was found that in a single dose of 1.5 mg/kg carminomycin inhibited the lymphoma colonies by 50 per cent. The maximum immunodepressive effect was observed when carminomycin was used in a single dose of 1.5 mg/kg 48 hours after the antigen stimulation. In this case the number of the antibody forming cells in DBA/2 mice with leukemia L-1210 was lower than that in CBA mice without leukemia.     

Suppressive effect of the blast cells of AKR strain mice on antibody formation in vivo and lymphocyte proliferation in vitro

Blast cells obtained from the "erythropoietic spleen" of FG-stimulated young mice and cells accumulating in the spleens of preleukemic AKR mice have a marked suppressive effect on spontaneous and mitogen-induced proliferation of young mouse splenocytes in vitro and suppress the development of humoral immune response in immunized recipients during syngeneic transfer in vivo. Some disturbances in erythron system in preleukemic AKR mice manifested in the accumulation of immature erythroid precursors which are suppressors of immunocompetent lymphocytes are suggested to be a pathogenetic link in the development of leukemia.     

Development of host-dependent high-grade tumor-specific immunity through a novel mechanism triggered by the Lyt-2+ tumor-specific T cell clone (K7L) that induces temporal growth of L1210 leukemia-K7L-variant

When a murine leukemia L1210-specific Lyt-2+ T cell clone, K7L, was injected i.p. into CD2F1 mice together with L1210, the normal growth of L1210 in the peritoneal cavity of the mice at the early stage (days 0 to 5) was strongly inhibited, but L1210 grew progressively at the middle-stage (days 5 to 10), and then was rejected at the late stage (days 10 to 20). The mice thus survived for long times (more than 60 days), whereas the normal control injected with L1210 alone died within 14 days. The L1210 that grew at the middle stage in mice initially inoculated with L1210 together with K7L was a K7L-insensitive (K7L-) variant. All of eight tumor clones established from L1210-K7L- by limiting dilution was insensitive to the antitumor activity of K7L, and this property of tumor clones was stable after repeated in vitro passage. The initial depression of the L1210 growth by K7L followed by growth and rejection of the variant L1210-K7L- by the host T cell activity was then found to prepare a strong, long-lasting (more than 3 mo) immunity to protect mice against the high-dose (10(7) cells per mouse) challenge of original L1210. Corresponding to this result, definite tumor (L1210)-specific cytotoxic T lymphocyte (CTL) activity against both variant and original L1210 targets was developed by antigen (L1210) restimulation in the culture of spleen cells from these mice, but was not increased to a detectable level before L1210-K7L- variant started to grow. It was suggested that the 1210-K7L- variant and the original L1210 should have the common tumor-specific antigen that was independent of the K7L-reactive antigen, and that original L1210, whose growth was retarded by K7L, primed the host with the common antigen to be enormously boosted by the subsequently growing L1210-K7L- variant.     

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Biologic Activity of a Nucleotide Conjugate Between Mitomycin C and Cytarabine Monophosphate

Abstract

A dual prodrug conjugate between the antimetabolite cytarabine monophosphate and the alkylating agent 2,7-diaminomitosene (derived from mitomycin C), cytaramycin, was synthesized and tested for antileukemic activity in sensitive and resistant tumors. The compound was active against parental L-1210, CCRF-CEM, HL-60 and K-562 leukemia cells but did not overcome resistance in sublines developed for (1) multidrug resistance (L-1210/MDR and K-562-R) or (2) for cytarabine resistance (CCRF-CEM/ARA-C and HL-60/ARA-C). Alkaline DNA elution tests demonstrate a predominance of strand breaking activity due to the cytarabine moiety, and a lesser degree of DNA crosslinking, due to the mitosene moiety. The conjugate was active in mice bearing P-388 leukemia (80% increased lifespan), but was not more effective than mitomycin C alone in mice bearing a cytarabine-resistant L-1210 cell line (38% to 31% increased lifespan). These findings suggest that mitomycin nucleotide conjugates do not overcome resistance to the parent antimetabolites.     

Kinetics and distribution of transplanted sarcoma JWS and leukemia L1210 cells by intravenous route

The aim of this work was to compare the distribution in organs of syngeneic mice sarcoma JWS and leukemia L-1210 cells previously labelled with sodium dichromate (Na2 51CrO4) and iododeoxyuridine (125IUDR) after i.v. transplantation. Also, the results obtained with both labels were compared. Both kinds of cells under study are trapped in lungs, but the number of trapped JWS cells is greater. L-1210 cells probably recirculate. The cells undergo extensive destruction during the first 3 days after transplantation. This kind of study requires the use of two markers: cytoplasmic and nuclear and careful analysis of radioactivity changes is also required to obtain proper conclusions.     

Effect of epidermal chalones on the growth of transplantable tumors]

The ethanolic extract of rat skin contains epidermal G1- and G2-chalones which having been added to cell suspension of transplantable squamous-cell cornified carcinoma of mice cervix, inhibits its growth in recipients by 72,6 per cent. The same extract failed to inhibit the growth of transplantable mouse tumours of other histogenesis (hepatoma 22a, leukemia L-1210 and sarcoma 180). When added to cell suspension of transplantable carcinoma of mouse skin which had been anaplized during a long period of transplantation (over 10 years) this extract inhibits its growth by 39,2 per cent only.     

Influence of T-lymphocyte deprivation on the antileukemic effect of bacillus calmette-guérin in vivo

Summary The effect in AKR mice of T-lymphocyte deprivation in vivo, obtained by adult thymectomy plus/minus whole-body irradiation and bone-marrow reconstitution, was studied in the growth of grafted leukemia cells obtained from spontaneous AKR leukemia. Both thymectomized mice and mice subjected to thymectomy, whole-body irradiation, and bone-marrow reconstitution (B) had a lower take-frequency of graft leukemia than conventional mice. Growth of graft leukemia was inhibited by BCG treatment both in thymectomized mice and in B mice. Concomitant with the increased inhibition of leukemia growth, an increased incidence of wasting-like disease was observed. In vitro cytotoxicity studies revealed that spleen lymphoid cells from nonleukemic mice were cytotoxic to AKR leukemia cells, to nonmalignant AKR fibroblasts, and to other nonmalignant cells with H-2^k haplotype. The activity of this self-directed cytotoxicity was most marked in AKR mice with wasting-like disease. The presence of autocytotoxic cells was frequently associated with a positive direct Coombs' test. Immunofluorescence studies showed, further, that the cytotoxic activity was independent of retrovirus antigens as tested by indirect immunofluorescence with anti-MuLV antibodies. Adult thymectomy of AKR mice confers an increased antitumoral immune potential, but also an increased risk of development of serious autoimmune disease.     

Purification and characterization of a novel cytotoxic substance from cell-free extract of Streptococcus pyogenes

A cytotoxic substance designated as streptococcal cytotoxic protein (SCP) was isolated from a cell-free extract of the Su strain of Streptococcus pyogenes possessing cytotoxic and antitumor activity. SCP was purified with a series of column chromatography and preparative PAGE to give a homogeneous single band as revealed by PAGE analysis. The purified SCP has a molecular mass of 165 kDa, composed of four 43 kDa subunits, and its pI is 4.3. SCP was sensitive to proteinases and was labile to heat and at acidic or alkaline pH. SCP showed inhibitory effects on the [3H]thymidine, [3H]uridine and [3H]leucine uptakes and on the growth of cells, and released 51Cr from cells when the protein was added to the cultures of Ehrlich ascites carcinoma (EAC), mouse mammary tumor (MM-2), leukemia (L-1210) and NIH-3T3 mammalian cells in vitro. SCP also showed an antitumor effect on EAC or MM-2 tumor-bearing mice but not on L-1210 tumor-bearing mice in vivo.     

CD4-CD8+ T lymphocytes mediate AKR/gross murine leukemia virus nonresponsiveness in moderately aged AKR.H-2b:Fv-1b mice.

Previously we reported that as AKR.H-2b:Fv-1b mice become older than 9 wk of age they begin to specifically lose the ability to generate anti-AKR/Gross murine leukemia virus (MuLV) CTL responses after immunization and in vitro restimulation with cells expressing AKR/Gross MuLV-encoded Ag. Interestingly, the frequency of virus-specific precursor cytotoxic T lymphocytes (CTL) observed in moderately-aged AKR.H-2b:Fv-1b mice was not substantially decreased from that found in their young responder counterparts. To further investigate the mechanism(s) responsible for the inability of moderately-aged AKR.H-2b:Fv-1b mice to mount AKR/Gross MuLV-specific CTL responses, adoptive transfer experiments were performed in the present study. Transferring splenocytes from moderately-aged AKR.H-2b:Fv-1b donors into young AKR.H-2b:Fv-1b recipients resulted in inhibition of AKR/Gross MuLV-specific CTL responsiveness. Anti-Thy-1.1 plus complement depletion of T cells from the donor cell population before adoptive transfer resulted in a near complete restoration of AKR/Gross MuLV responsiveness of young recipient AKR.H-2b:Fv-1b mice suggesting that the inhibition observed in moderately aged mice was mediated by T lymphocytes. Additional experiments using depletion of T subsets before cell transfer demonstrated that inhibition of AKR/Gross MuLV-specific CTL responsiveness was mediated by a CD4-CD8+ T lymphocyte.     

Maternally transmitted resistance to lymphoma development in mice of reciprocal crosses of the RF/J and AKR/J strains

Females but not males of the low-lymphoma RF/J strain transmit a non-Mendelian factor which suppresses the development of lymphoma in F1 crosses with mice of the high-lymphoma AKR/J strain. Suppression of lymphoma was also evident in the first backcross generation to the parental AKR strain, but only when (RF female x AKR male)F1 mice had been the female parent. This "maternal resistance factor" was transmitted independently of the dominant, lymphoma-suppressing Fv-1n allele transmitted by both males and females of the RF strain, but the suppressive capacities of the two factors appeared to be additive. In this cross, F1 progeny of RF females also showed marked suppression of ecotropic murine leukemia virus expression by comparison with mice of the reciprocal F1 cross, but this suppression of virus expression was not detected in the lymphoma-suppressed AKR backcross population. The observation of lymphoma suppression in the absence of ectropic virus suppression in mice of the (RF X AKR)F1 female x AKR male backross generation indicates a qualitative or quantitative difference in the determination of these two effects.     

Genetic control of the induction of cytolytic T lymphocyte responses to AKR/Gross viral leukemias. II. Negative control by the Fv-1 locus in AKR mice of responder H-2b haplotype

To assess whether the presence of a responder H-2b haplotype would be sufficient to allow mice of nonresponder "high leukemic" phenotype to generate syngeneic anti-AKR/Gross virus cytolytic T lymphocytes (CTL), the AKR.H-2b strain was examined. Although capable of mounting vigorous apparent anti-minor histocompatibility-specific CTL responses, AKR.H-2b mice failed to produce anti-viral CTL after a variety of stimulation protocols. In contrast, the "doubly congenic" AKR.H-2b:Fv-1b strain was able to respond with substantial levels of H-2-restricted anti-AKR/Gross virus CTL activity. These results indicated that Fv-1n alleles could exert negative epistatic control over responder H-2b-encoded gene(s). Because the B6.Fv-1n congenic was also able to generate anti-viral CTL indistinguishable from the prototype B6 strain, however, it was apparent that other genes of AKR background were required for the Fv-1n-mediated inhibition in AKR.H-2b mice. The mechanism by which Fv-1 intereacted with other genes to override positive H-2b control appeared to be related to the expression of the CTL-defined, virus-associated antigens by normal AKR.H-2b cells. Thus, AKR.H-2b spleen cells but not thymus cells were able to stimulate the production of B6 anti-AKR/Gross virus CTL and were recognized as target cells by such anti-viral CTL. In contrast, both spleen cells and thymocytes from AKR.H-2b:Fv-1b mice were negative when tested as stimulator or target cells in these assays. In addition, AKR.H-2b but not AKR.H-2b:Fv-1b spleen cells were shown to display serologically defined gp70 determinants and the Gross cell surface antigen. Taking these data together, it appeared that the inhibition of anti-viral CTL responsiveness might be due to tolerance induced by the cell surface expression of virus-associated antigens by normal AKR.H-2b cells. Widespread display of viral antigens, in turn, may have been due to the permissive effects of Fv-1n on the spread of the early arising N-ecotropic, endogenous AKR leukemia virus controlled by other background genes. In this context, the implications of the multi-gene control of anti-AKR/Gross virus CTL production are discussed with respect to the induction of spontaneous leukemia in the high incidence AKR strain.     

Increase in the sensitivity of leukemia cells, incubated in interferon, to the effect of immune serum]

L-1210 AND P-388 leukemic cells were incubated in three types of interferon, i.e. L-cell interferon and two types of lymphocyte interferon (induced in the lymph node lymphocytes of intact mice and the lymphocytes obtained on the 10th day after intraperitoneal injections of 5-10(7) L-1210 cells). "False" interferon obtained by the method analogous to that of obtaining L-cell interferon, excluding the viral induction, was used for control. Cells incubated in interferon proved to be more sensitive to the action of the cytotoxic antibodies than those treated with "false" interferon. Treatment of the cells with lymphocytic interferon induced on the lymphocytes of immune mice increased their sensitivity even more in comparison with the cells treated with interferon obtained from intact mice.     

Induction of anti-AKR/gross virus cytolytic T lymphocytes in AKR.H-2b:Fv-1b congenic mice: age-dependent conversion to a nonresponder phenotype

Previously, we reported that the generation of cytolytic T lymphocytes (CTL) specific for syngeneic tumors induced by AKR/Gross leukemia viruses was under multi-gene control. Thus, although carrying the required immune response gene(s) encoded by the H-2b haplotype and characteristic of responder strains such as C57BL/6, AKR.H-2b congenic mice failed to mount antiviral CTL responses. Young adult AKR.H-2b:Fv-1b "doubly congenic" mice, however, were able to generate specific anti-AKR/Gross virus CTL activity. These results demonstrated that the positive effect of MHC-encoded immune response gene control could be overcome by the action of the Fv-1n allele. The responder status of the B6.Fv-1n congenic, however, indicated that this Fv-1n-mediated inhibition was dependent on the interaction of Fv-1n with another gene(s) encoded by the AKR background. The results of experiments performed with AKXL recombinant inbred mice further suggested that a single additional genetic locus, encoding the Akv-1 provirus, was necessary along with Fv-1n to cause inhibition of antiviral CTL generation. Here we show that the responsiveness of AKR.H-2b:Fv-1b mice is dependent on their age. Thus, with moderate aging these doubly congenic mice converted to a nonresponder status with respect to anti-AKR/Gross virus CTL production: 85% of mice less than or equal to 9 wk of age responded compared with 0% of mice greater than 9 wk old. As with nonresponder AKR.H-2b mice, an inverse correlation was observed between CTL responsiveness and the expression of CTL-defined viral antigens by normal cells. Namely, spleen cells from young AKR.H-2b:Fv-1b mice showed little or no expression of such viral antigens, whereas with moderate aging there was a steady increase in their display. These results are discussed with reference to possible mechanisms of unresponsiveness of AKR.H-2b vs moderately aged AKR.H-2b:Fv-1b mice, and with respect to the utility of this system as a model for naturally occurring retrovirus infections and the interactions of retroviruses with the immune system.     

Deficiency in early development of the thymus-dependent cells in irradiation chimeras attributable to recipient's environment.

Allogeneic bone marrow chimeras were prepared using reciprocal combinations of AKR and C3H mice. When C3H mice were recipients, the number of thymocytes recoverable from such chimeras (C3H recipient chimeras) was small as compared with that from chimeras for which AKR mice were used as recipients (AKR recipient chimeras) regardless of donor strain. The thymocytes from C3H recipient chimeras showed a profound deficiency in generating proliferative responses to stimulation by anti-CD3 mAb (2C11) or anti-TCR (alpha, beta) mAb (H57-597), even though the expression of CD3 and TCR molecules fell within the same range as that in AKR recipient chimeras. Furthermore, after stimulation with immobilized 2C11, the proportion of IL-2R+ cells in the thymocytes from C3H recipient chimeras was much less than that in AKR recipient chimeras. However, no significant difference in proliferative responses to 2C11 plus PMA, in influx of Ca2+ after stimulation with 2C11 or IL-2 production in response to 2C11 plus PMA or PMA plus A23187 was demonstrated between C3H and AKR recipient chimeras. These findings suggest that the thymocytes from C3H recipient chimeras have a deficiency in the signal transduction system as compared with chimeras for which AKR mice are the recipients. The thymic stromal component involved in this difference in the C3H recipient chimeras is discussed.     

Utilization of chromosomal markers in cancer research]

The mouse is an unsuitable species for cytogenetical studies to the extent that it has 40 acrocentric chromosomes and the only criterion which could be used to differentiate them is size. We envisaged using in the case of cell grafts donors or recipients of different sex. This technique has, however, been used to a limited extent. Among the other markers which have been utilized, T6T6 of CBA mice must be mentioned. The discovery in 1966 by Léonard and Dekundt of the presence in AKR mice of fusions of the Robertson type (between chromosomes 6 and 15) has generated new interest in experimental work based on the utilisation of chromosome markers. Being interested in the mechanisms of radio-induced leukemia, the authors described how they have introduced the chromosome marker of AKR mice into the C57B1 strain which is very sensitive to the induction of radio-induced leukemias.     

Disruption of DNA synthesis and structure of mouse L1210 leukemia cells, sensitive and resistant to 1-methyl-1 nitrosourea and 1,3-bis(2-chloroethyl)-1-nitrosourea in vivo

Leukemia L1210 cells with acquired resistance to 1-methyl-1-nitrosourea (MNU) (L1210/MNU) and 1.3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (L1210/BCNU) were developed from leukemia L1210 cells sensitive to these drugs (L1210/0). The modal chromosome number of leukemia L1210/MNU and L1210/BCNU cells increases from 40 (L1210/0) to 41. It was shown that in leukemia L1210/MNU cells the inhibition of DNA synthesis after MNU administration in a therapeutic dose (80 mg/kg) is lasted within 24 hours, while that in leukemia L1210/0 cell--within 96 hours. After administration of BCNU (20 mg/kg) inhibition of DNA synthesis in leukemia L1210/BCNU cells reached of 50% of control in comparison with practically complete inhibition of DNA synthesis in leukemia L1210/0 cells. Centrifugation on alkaline sucrose density gradients revealed no differences in the rate of sedimentation of leukemia L1210/0, L1210/MNU and L1210/BCNU cell lysates. After 1 hour treatment with MNU of mice bearing L1210/MNU and L1210/0 leukemia cells single-strand breaks in DNA were determined. After 4 hours these strand-breaks retained in leukemia L1210/0 cells, but were eliminated in leukemia L1210/MNU cells. Administration of BCNU to mice with leukemia L1210/0 and L1210/BCNU cells resulted in both cases in the production of DNA aggregates. There is no complete cross-resistance between MNU and BCNU which allows a substitution of these drugs providing for the increase in their therapeutic efficiency.     

Detection of antigens of endogenous viruses of the C-type during mouse development]

Embryos of the 12th-20th day of gestation, newborn and adult AKR and BALB/c mice were investigated for the presence of mouse C-type virus major structural p30 protein (gs-1) and Gross leukemia virus type-specific antigen AGLV) by means of radioimmunodiffusion with test systems. The p30 protein was distinctly determined from the 12th day of intrauterine development in both mouse lines; it persisted in the embryo tissues until birth and was detectable also in the AKR and BALB/c mouse tissues from the first days of postnatal development and during the whole life. AGLV was not revealed in BALB/c and AKR embryos and in adult BALB/c mice; however it was found in the AKR newborn mice since the 1st-2nd day after birth. Basing on these data a conclusion was drawn that p30 protein and AGLV were expressed independently according to the radioimmuno-diffusion method sensitivity.     

Distribution and elimination of transplanted sarcoma JWS and leukemia L1210 cells in allogeneic recipients--inbred C57BL mice by intravenous route]

The distribution of neoplastic--JWS sarcoma and lymphatic leukemia L-1210 cells after intravenous injection into allogeneic recipients is presented. Cells were labelled with two labels: cytoplasmic (sodium chromate-51CR) and nuclear (iododeoxyuridine-125IUDR). Radioactivity of blood, lungs, liver, spleen and kidneys was measured 90 minutes and 24 hours after cell transplantation. The pattern of cell trapping, destruction and elimination from the circulation was characteristic of cell line injected. Destruction and elimination processed faster in allogeneic system than in syngeneic one.