Short communication: Antiproliferative effect of 8 different Lactobacillus strains on K562 cells.

Some strains of Lactobacillus genus have antiproliferative activities against cancer cells. However, until now, the exact effector molecules of Lactobacillus strains with anticancer activity have not been identified. The aim of the present study was to explore which fraction of the Lactobacillus cells exerts the highest antiproliferative effect. For this purpose, the heat-killed bacterial cells, bacterial cell wall extract, and genomic DNA of 8 Lactobacillus strains were prepared to assess their antiproliferative activities against human myeloid leukemia cell lines K562. The heat-killed bacterial cells of the 8 lactobacilli strains exerted antiproliferative effect on K562 cells, and the inhibition rates exerted by the heat-killed bacterial cells of the strains G15AL, M5AL, SB31AL, SB5AL, and T3AL were significantly higher than those exerted by the cell walls and genomic DNA of the strains. The bacterial DNA of G15AL exerted higher antiproliferative effect on K562 cells. The exact effector molecules and the effect mechanism of the strains should be further explored for the application of these strains as probiotic strains or bioactive probiotic molecules.

Modulation of rhodamine 123 uptake by nigericin in sensitive and multidrug resistant leukemic cells.

We have investigated the effect of the ionophore nigericin (NIG) in multidrug resistant (MDR) cells, using intracellular accumulation of the fluorescent dye rhodamine 123 (R123). NIG increased the accumulation of R123 in half of the murine MDR RFLC3 population but not in the human MDR CEM/VLB 100 cells. Co-treatment of RFLC3 with NIG plus verapamil showed additive effect on the accumulation of R123. The increase in R123 accumulation observed in RFLC3 was not the consequence of a direct effect of NIG on P-glycoprotein and was accompanied by a redistribution of the dye throughout the cell and a high cytotoxicity, which prevents the use of NIG as a resistance modulating agent.

Contrasting effects from a single major histocompatibility complex class II molecule (H-2E) in recovery from Friend virus leukemia.

Resistance to erythroleukemia induced by infection with the Friend virus complex (FV) has been mapped to several genes residing both within and outside the murine major histocompatibility complex (MHC). MHC genes located in the A, D, and Qa/Tla regions of the murine H-2 complex have been shown to affect disease resistance through their capacity to regulate various aspects of the host immune response to viral antigens. This study establishes H-2E as the fourth MHC locus controlling immunological resistance to FV. Our investigation into the role of H-2E molecules revealed two distinct and opposite effects on recovery from Friend disease. H-2b/b mice normally lack a functional E gene product and are resistant to high doses of FV. The expression of H-2E molecules in H-2 recombinant or transgenic mice of this genotype resulted in a significant decrease in spontaneous recovery from FV-induced leukemia. In contrast, H-2E expression also appeared to influence recovery from Friend disease in a positive manner, since blocking these molecules with anti-E antibodies in vivo significantly decreased recovery from Friend disease. The data indicate that the positive effects of H-2E molecules derive from their function as restriction elements for helper T-cell recognition of the viral envelope glycoprotein, and we postulate that the negative effects are due to H-2E-dependent deletion in the T-cell repertoire during development.

Therapeutic effects of D-aspartic acid beta-hydroxamate (DAH) on Friend erythroleukemia.

D-aspartic acid beta-hydroxamate (DAH), an aspartic acid analogue, exerts anti-tumoral activity against murine leukemia L5178Y both in vitro and in vivo. We show here that DAH displays activity against Friend leukemia cells (FLC) in vitro: a concentration of 2 mM results in a total inhibition of cell growth. DAH is also active in vivo against Friend virus (FV-P)-induced erythroleukemia. Treatment with DAH, given for 95 days as a single daily i.p. injection to DBA/2 mice 3 days following FV-P inoculation, induced a marked increase of 212% in the mean survival time (MST) of treated animals. Since FV-P-induced erythroleukemia is characterized by the proliferation of mature erythroid precursors, we examined the effect of DAH treatment on erythroid colony-forming cells (CFU-E) and observed that the number of CFU-E per spleen was 30 times lower in DAH-treated mice than in the controls. To gain further insight into the early effects of DAH treatment on the early phase of Friend disease, we examined the effects of short DAH treatment on spleen size, hematocrit and viremia in FV-P-infected mice. DAH treatment initiated 3 days post infection (p.i.) inhibited splenomegaly, prevented virus-induced polycythemia, and reduced serum viremia. Late DAH treatment (18 days p.i.) induced regression of FVP-induced disease as evidenced by reduction of spleen weight.

Retroviral capture of c-erbB proto-oncogene sequences: rapid evolution of distinct viral genomes carrying mutant v-erbB genes with different transforming capacities.

The evolution of oncogene-transducing retroviruses was followed by studying the genomes of five new, erbB carrying retroviruses. These viruses, isolated from cells of one chicken infected with Rous Associated virus 1 (RAV-1), had captured c-erbB sequences as a consequence of RAV-1 integration into the host genome. Their genome structures were distinct; however, their v-erbB genes had sustained identical 5' and 3' deletions and the v-erbB-env junctions were identical at the nucleotide level. The results therefore strongly suggest that all five viruses originate from the same capture event. Sequence analyses of the v-erbB genes from three of these viruses revealed that one of them had undergone no further mutation and lacked detectable capacity to transform cells, therefore probably representing an 'early' form of transducing virus. The two other v-erbB genes contained distinct mutations and differed in their potential to induce fibroblast- and erythroblast transformation; they therefore probably represent later derivatives of the virus that captured the erbB oncogene. The data suggest that the initial retrovirus rapidly underwent many alterations after capture of c-erbB sequences, already in the RAV-1 infected bird as well as during subsequent in vitro isolation procedures. The changes involve both major rearrangements of the genome as well as point mutations that activated the erbB oncogene.

Receptor-targeted transfection using stable maleimido-transferrin/thio-poly-L-lysine conjugates.

Cell surface ligand-receptor systems provide favorable routes for DNA transfection due to target cell specificity, transfer efficiency, and low toxicity. Using the transferrin receptor system as a model, an approach to transfection is developed here within which involves the complexing of DNA to stable maleimido-transferrin/thio-poly-L-lysine conjugates. These studies establish the importance of precise stoichiometry for activity of ligand:poly-L-lysine conjugates, as well as a chemistry for their controlled conjugation. Also considered quantitatively are effects of the following related parameters on the efficiency of receptor-mediated transfection: lysine polymer length, conjugate concentration, DNA:conjugate ratio, and treatment of target cells with chloroquine and desferrioxamine. Compared directly to standard procedures (electroporation, modified DEAE-dextran, lipofection, and modified Ca2PO4 protocols), transfection via this transferrin receptor-mediated system was > or = 10-fold more efficient, and essentially nontoxic to erythroleukemic F-MEL and J2E cells. Following transfection these cells retained the physiological capacity to undergo induced differentiation in response to dimethyl sulfoxide (F-MEL cells), or to erythropoietin (J2E cells), the natural hormonal regulator of erythropoiesis. Thus, an optimized approach to transferrin receptor-mediated transfection is developed which should be widely applicable for alternate cells and ligand-receptor systems both in vitro and in vivo.

The role of gp55 N-glycosylation in pathogenesis of Friend spleen focus-forming virus.

The product of the envelope gene (gp55) of Friend spleen focus-forming virus is responsible for the acute form of erythroleukaemia caused by this virus. In order to investigate the role that the four known N-linked carbohydrate side-chains of gp55 play in pathogenesis, we have inactivated the four N-glycosylation signals by mutating the asparagine residues of these four sites into serine. When glycosylation sites 1 and/or 2 were altered, the viruses remained fully pathogenic. However, mutation at either of glycosylation sites 3 or 4 rendered the virus apathogenic, independent of mutations at other sites. Furthermore, when site 3 was changed, a new product appeared which seemed to have acquired a carbohydrate chain at a position normally not glycosylated, presumably at position Asn378.

A low oncogenic variant of Friend murine leukemia virus with strong immunosuppressive properties.

Friend leukemia complex (FLC) is known to induce immunosuppression but the use of FLC in studies of immune cells function is disadvantageous since the immunosuppression always is accompanied by an acute erythroleukemia. To obtain immunosuppressive variants of FLC with reduced leukemogenic potential, we isolated T-helper cells from FLC infected mice, and passed lysates of the cells to recipient uninfected mice. A group of these mice developed a condition distinct from the disease induced by FLC. A viral stock prepared from these mice, designated Fd-MIV for friend derived murine immunodeficiency virus, induced a profound suppression of the primary antibody response without acute transformation in adult NMRI mice. Terminally a wasting disease with weight loss, atrophy of the thymus and lymph nodes and renal disease was observed in some mice. Analysis of viral DNA and RNA from infected NIH 3T3 cells showed that Fd-MIV contained at least two viral components, a 8.4 kb friend murine leukemia virus (F-MuLV) and a 7.4 kb mink cell focus (MCF)/xenotropic virus related genome. The 7.4 kb genome was not detected in Fd-MIV infected, immunocompromised mice indicating that the 8.4 kb genome might be responsible for the disease.

Thrombocytopenia in a retrovirally-induced murine erythroleukemia.

A variant strain of Rauscher leukemia virus (RLV-A) obtained from a transplantable murine monomyelocytic leukemia causes a disease characterized by frank anemia, wasting, hepatosplenomegaly and erythroblastosis. The involvement of platelets in this disease are reported here. The RLV-A induced a severe thrombocytopenia (25 percent of control level) at the terminal stage of disease. This thrombocytopenia was not associated with disseminated intravascular coagulopathy since the prothrombin times were always within normal limits. The partial thromboplastin time was elevated in the terminal stages of disease and was found to be associated with factor deficiencies, possibly owing to the presence of anti-factor antibodies, in the intrinsic coagulation pathway, especially factor VIII. Further, splenectomy did not abolish the thrombocytopenia, since splenectomized, virally infected animals also developed severe thrombocytopenia (29 percent of control levels). The ensuing splenomegaly during progression of disease was not the cause of the thrombocytopenia. A physiological response to the severe thrombocytopenia was the production of larger size platelets. At terminal stages of the disease, platelet volume increased to 4.2 mu 3 (normal is 3.0 mu 3). An increase in platelet volume was also observed in splenectomized, virally infected animals. Electron microscopy indicated that these circulating platelets contained c-type viral particles. Viral infection was associated with decreased life span of circulating platelets, as measured by 75Se-methionine at mid and terminal stages of the disease. Our results suggest that direct viral infection of platelets and/or megakaryocytes with subsequent cell lysis is a possible cause of the observed thrombocytopenia observed in RLVA-induced disease and may also occur in other retrovirally-induced diseases.

A carboxy-terminal mutant spleen focus-forming virus (SFFV) envelope glycoprotein is transport-competent, but non-leukemogenic.

The Friend spleen focus-forming virus (F-SFFV) codes for a transport-defective leukemogenic envelope glycoprotein designated as gp52. We have previously shown that the external domain of gp52 carries the determinants responsible for its transport defect. Consistent with this idea, truncated gp52 molecules that lack a hydrophobic membrane anchor were transport-defective, and were not secreted from cells. In this report, we describe the construction of a mutant SFFV envelope gene that codes for altered gp52 molecules in which the carboxyl-terminal hydrophobic residues are replaced with exogenous hydrophilic residues encoded by the vector-derived sequences. The mutant env gene was expressed using the retroviral expression vector, pLXSN, and the mutant envelope protein was found to be transport-competent, and efficiently secreted from the cells. However, M-MuLV pseudotypes of the retroviral vectors expressing the mutant genome were found to be non-leukemogenic in mice.

Friend murine leukaemia virus is integrated at a common site in most primary spleen tumours of erythroleukaemic animals.

A common proviral integration site was identified and characterized in erythroleukaemias induced by Friend murine leukaemia virus (F-MuLV). Using inverse polymerase chain reaction, we found a proviral integration site common to at least 90% of 20 primary tumours tested. This site was identical to Fli-1, a locus recently reported by others to be rearranged in 75% (9/12) of cell lines established from spleens of erythroleukaemic mice and to code for a member of the ets gene family. Our data suggest that about half of the F-MuLV-induced erythroleukaemias contained more than one cell clone with a proviral integration in Fli-1, with different individual integration sites within Fli-1 in each cell clone. All proviruses were found to be integrated in the same transcriptional orientation with respect to flanking cellular DNA. We discuss these findings in relation to multistage models of neoplasm.

Changes in pyridine and adenine nucleotide levels in Friend erythroleukaemia cells during growth and differentiation.

Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.

Nuclear protein kinase C activity is decreased in Friend erythroleukemia cells induced to differentiate.

Although it is well known that protein kinase C (PKC) is an important signaling molecule in Friend erythroleukemia cells it is not clear what role PKC may play in either regulated or unregulated erythroid cell proliferation and differentiation. The purpose of this study was to test the hypothesis that a decrease in nuclear PKC activity is associated with the induction of differentiation in Friend erythroleukemia cells. The effects of staurosporine, a selective inhibitor of PKC, and the tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate, an activator of PKC, on Friend cell proliferation and differentiation were examined. Neither the inhibitor nor the activator of PKC affected proliferation at 96 h as measured by [3H]thymidine incorporation, but both compounds inhibited cell differentiation. In addition, nuclear PKC activity was highest in untreated and in tumor promoter-treated cells that were not differentiated, and it was lowest in cells induced to differentiate with hexamethylene bisacetamide or dimethylsulfoxide. It is concluded that nuclear PKC activity is essential for Friend erythroleukemia cell proliferation, and that a decrease in enzyme activity within the nucleus is associated with differentiation.

Sequences present in a small region of the AKV virus envelope gene determine the efficiency with which pseudotyped spleen focus-forming virus infects erythroid target cells.

Induction of erythroleukemia in mice by the replication-defective spleen focus-forming virus (SFFV) relies on the presence of a helper virus to deliver the SFFV genome to erythroid target cells. Pseudotyping studies with different ecotropic murine leukemia viruses (MuLV) have shown that SFFV pseudotyped with Akv, the endogenous ecotropic virus of AKR mice, inefficiently gives rise to virus-induced erythroid bursts (vBFU-E) in vitro and fails to cause erythroleukemia in mice when compared to SFFV pseudotyped with Friend or Moloney MuLV. In order to locate the region(s) of the Akv genome responsible for its inability to act as a helper for SFFV, six different Moloney MuLV chimeras containing Akv envelope sequence substitutions were constructed. Virions with the chimeric envelopes were used to pseudotype SFFV and the complexes were analyzed for their ability to induce vBFU-E in vitro and erythroleukemia in mice. SFFV preparations pseudotyped with three of the constructs containing chimeric envelope genes efficiently gave rise to vBFU-E as did SFFV pseudotyped with Moloney MuLV. SFFV pseudotypes generated from the other three constructs, which all share a common 304-bp region located near the center of the Akv gp70 coding region, and Akv gave rise to very few vBFU-E. However, all SFFV preparations, with the exception of SFFV pseudotyped with Akv, induced erythroleukemia in mice. The results suggest that specific sequences present in the envelope gene of Akv are responsible for the inefficiency of the virus to infect erythroid target cells for SFFV, but additional Akv sequences outside those used in this study affect the ability of the Akv/SFFV virus complex to cause erythroleukemia in mice.

p53 transgenic mice: accelerated erythroleukemia induction by Friend virus.

Mutations in the p53 tumor-suppressor gene have been implicated in the pathogenesis of a significant proportion of human cancers and in a dominantly inherited familial cancer syndrome (Li-Fraumeni syndrome). Frequent rearrangements and point mutations have also been detected in the p53 gene in the murine erythroleukemias induced by Friend leukemia virus. We have previously reported that transgenic mice overproducing a mutated p53 protein are predisposed to the development of lung carcinomas, bone and soft-tissue sarcomas, as well as lymphoid malignancies. Here we report that p53 transgenic mice infected with the polycythemia-inducing strain of Friend virus (FV-P) progress to the late stage of erythroleukemia more rapidly than do normal mice. In addition, Friend leukemic cell lines derived from p53 transgenic mice overproduce mutant p53 protein and show a high frequency of rearrangement of the ets-related Spi-1 oncogene, as previously reported in Friend cell lines derived from non-transgenic animals. These results suggest that the same genetic changes involved in the evolution of Friend leukemia in normal mice are also required in mice with an inherited predisposition to cancer. The data also indicate that p53 transgenic mice provide an animal model in which to analyse the role that genetic and environmental factors play in influencing cancer predisposition.

Substitution of leucine for isoleucine in a sequence highly conserved among retroviral envelope surface glycoproteins attenuates the lytic effect of the Friend murine leukemia virus.

Friend murine leukemia virus is a replication-competent retrovirus that contains no oncogene and that exerts lytic and leukemogenic properties. Thus, newborn mice inoculated with Friend murine leukemia virus develop severe early hemolytic anemia before appearance of erythroleukemia. To identify the retroviral determinants regulating these effects, we used chimeric infectious constructions and site-directed point mutations between a virulent Friend murine leukemia virus strain and a naturally occurring variant attenuated in lytic and leukemogenic effects. We found that severe hemolytic anemia was always associated with higher numbers of blood reticulocytes with budding retroviral particles. Furthermore, a remarkably conservative leucine to isoleucine change in the extracellular SU component of the retroviral envelope was sufficient to attenuate this lytic effect. Also, this leucine at position 348 of the envelope precursor protein was located within the only stretch of five amino acids that is conserved in the extracellular SU component of all murine, feline, and primate type C and type D retroviral envelopes. This observation suggested an important structural function for this yet undescribed conserved sequence of the envelope. Lastly, we observed that lytic and leukemogenic effects were attenuated by a deletion of a second repeat in the transcriptional enhancer region of the viral long terminal repeats of the variant strain.

Erythroleukemia induction by Friend murine leukemia virus: insertional activation of a new member of the ets gene family, Fli-1, closely linked to c-ets-1.

The retroviral integration site Fli-1 is rearranged in 75% of the erythroleukemia cell clones induced by Friend murine leukemia virus (F-MuLV), whereas Spi-1/PU.1, a member of the ets family of DNA-binding proteins, is rearranged in 95% of the erythroleukemias induced by Friend spleen focus-forming virus (SFFV). To determine the transcriptional domain defined by Fli-1, we have isolated a cDNA clone that is highly expressed only in erythroleukemia cell lines with Fli-1 rearrangements. The protein sequence of this cDNA is very similar to Erg2, another member of the ets gene family. The hydrophilic carboxy-terminal end of the Fli-1 cDNA shares significant sequence similarity to the DNA-binding ETS domain found in all members of the ets family. PFGE analysis localized Fli-1 within 240 kb of the ets-1 proto-oncogene on mouse chromosome 9 and human chromosome 11q23, suggesting that ets-1 and Fli-1 arose from a common ancestral gene by gene duplication. The involvement of the murine Fli-1, Spi-1, and avian v-ets genes in erythroleukemia induction suggests that activation of ets gene family members plays an important role in the progression of these multistage malignancies.

Influence of the site of tumor growth on the capacity of a low tumorigenic line of Friend erythroleukemia cells to differentiate.

Friend erythroleukemia cells (FLC) passaged in mice are highly tumorigenic and multiply extensively in the livers of suckling DBA/2 mice without differentiating. In contrast, in vitro passaged FLCs injected intravenously were of low tumorigenicity, multiplied to a limited extent in the livers of suckling mice, and underwent marked differentiation from the proerythroblast to the orthochromatic erythroblast stage in the liver. The presence of characteristic C-type virions budding from the cell surface in various stages of erythroid differentiation served as a marker of the injected FLCs. When the same in vitro passaged FLCs that differentiated in the liver were injected subcutaneously in suckling mice, they formed large subcutaneous tumors consisting of sheets of undifferentiated tumor cells. It is concluded that the tumorigenicity of FLCs depended on the site of tumor growth and that there is an inverse correlation between the tumorigenic capacity and the capacity to differentiate.