Search

Five different types of protein kinase acivities have been evaluated in cell lines from murine lymphomas induced by Abelson leukemia virus (A-MuLV), whose oncogene codes for a tyrosine protein kinase. Such activities were compared with those of normal cells and of cells transformed by Moloney leukamia virus (M-MuLV), lacking oncogene sequences in its genome. While cAMP-dependent protein kinase and casein kinase-1 do not undergo significant changes, casein kinase-2 rises in both A-MuLV and M-MuLV infected lymphocytes, becoming largely associated with the particulate fraction of transformed cells. Protein kinase-C on the other hand is unchanged in M-MuLV transformed cells but it undergoes a 2–3-fold increment in both soluble and particulate fractions of A-MuLV transformed lymphocytes, which also display high tyrosine protein kinase activity.

In cultures of hamster fibroblasts (BHK cell line) treated with potassium dichromate (K 2 Cr 2 O 7 ) nucleic acid and protein syntheses are differentially inhibited, and nucleoside uptake into the intracellular pool is characterized by a stimulation phase followed by an inhibition phase. Different patterns are observed for the uptake of each ribo- and deoxyribonucleoside, pyrimidine nucleoside (particularly deoxycytidine) uptake reaching the highest stimulation level. Kinetics of thymidine and deoxycytidine initial uptake at different exogenous nucleoside concentrations show that Ka 2 Cr 2 O 2 affects both simple and facilitated diffusion of nucleosides. The time course of thymidine and deoxycytidine pool saturation suggests however that the effects of K 2 Cr 2 O 7 on plasma membrane permeability are partially counterbalanced by modifications of pool size deriving from the concomitant alteration of steps of nucleoside metabolism separate from nucleoside uptake.

We studied the DNA replicon size in BALB/c and BALB/Mo mouse lymphocytes by the method of bromodeoxyuridine photolysis. After treatment of the BALB/Mo lymphocytes in vitro with mitomycin C, the average DNA replicon size appeared to be significantly smaller than that observed in BALB/c lymphocytes treated similarly. In these conditions as increased susceptibility to SCE induction in BALB/Mo lymphocytes had been observed. In the presence of both mitomycin C and cordycepin (an antiviral drug), both the DNA replicon size and the SCE frequency returned to normal values.

Abelson murine leukemia virus (A-MuLV) is a replication-defective retrovirus capable of rapidly inducing leukemia in mice as well as of transforming in vitro mouse bone marrow cells and fibroblasts [1, 2, 14]; the A-MuLV was derived by passage in vivo of the replication-competent Moloney leukemia virus (M-MuLV) and its genome codes for a single polypeptide which is a hybrid molecule containing a portion of the parental M-MuLV genome (gag gene) and a portion of the cellular gene termed abl [18]. This protein, which varies in size from 160 to 90 kilodaltons, depending on the specific A-MuLV strain, is associated mainly with the detergent-insoluble cell fraction and possesses a tyrosine kinase activity [4, 15, 19]. In this regard, the A-MuLV protein resembles the pp60src encoded by the Rous sarcoma virus (RSV) [10] and the protein kinases encoded by other retroviruses such as the feline sarcoma virus (FeSV) [3, 17] and the Fujinami sarcoma virus (FuSV) [9].

Cell lines derived from A-MuLV induced thymic lymphomas in BALB/c and C57BL/6 mice were analysed for their in vivo and in vitro potential of growth. Despite their immunogenicity, cell lines of BALB/c origin readily grew in syngeneic recipients. On the contrary, all cell lines of C57BL/6 origin failed to grow in immunocompetent hosts even though they were able to form tumours in immunosuppressed syngeneic mice. Among C57BL/6 lymphoma cells progression toward a more malignant phenotype was observed in TB6-3 cells, and in their derived clones, after several in vitro passages. This event was accompanied by the in vitro loss of requirement for exogenous growth factor(s) when tumorigenic TB6-3 cells were plated at high density. Moreover, culture medium from fully malignant TB-3 cells was mitogenic for mature T-lymphoma cells suggesting the involvement of an autocrine mechanism in the control of cell proliferation. Apparently, the viral oncogene (v-abl) is not directly involved in malignant progression since no differences between nontumorigenic and tumorigenic cells could be detected in A-MuLV integration patterns, v-abl specific mRNA expression, and P160gag-abl production. Images Figure 1 Figure 2 Figure 3

BALB/Mo mice lymphocytes, carrying endogenous Moloney murine leukaemia virus (M-MuLV), show significantly higher in vitro baseline frequencies of sister chromatid exchange (SCE) than lymphocytes from control (M-MuLV free) BALB/c mice. In vitro treatment of lymphocytes with cordycepin (10 micrograms/ml), an antiviral substance, lowers the level of SCE in BALB/Mo cells to the same value of BALB/c cells. The drug also reduces the higher sensitivity of BALB/Mo compared to BALB/c lymphocytes to the induction of SCE by mitomycin C (MMC) administered either in vitro (3 x 10(-8), 10(-7) M) or in vivo (0.3, 3 mg/kg). BALB/Mo lymphocytes treated in vivo with a high dose of MMC (10 mg/kg) show reduced susceptibility to the induction of SCE but increased frequencies of chromatid breaks and micronuclei. In this situation, cordycepin increases the level of SCE in BALB/Mo lymphocytes to exactly the level seen in BALB/c cells, but it does not affect the frequency of chromosomal aberrations. Since cordycepin is known to inhibit poly(A) synthesis, thus blocking RNA maturation, it is suggested that M-MuLV proviral integration is not the sole factor responsible for the altered susceptibility of BALB/Mo lymphocytes to SCE induction, but that it is most likely viral gene expression that is needed for this effect to occur. On the contrary, the high susceptibility of BALB/Mo lymphocytes to the induction of chromatid aberrations by a high dose of MMC administered in vivo seems to be independent of viral maturation.

Phosphotyrosine antibodies were employed to immunodecorate and immunoprecipitate proteins phosphorylated at tyrosine residues in cells transformed by Abelson murine leukemia virus (A-MuLV). In pre-B and pre-T lymphoma cells transformed by A-MuLV, the major phosphotyrosine-containing protein has an MW of 160 kDa and shares immunologically detectable sequences with the v-abl oncogene product. Moreover, two different proteins of approximately 100 and 68 kDa, heavily phosphorylated at tyrosine, were identified. Lack of immunological cross-reactivity with viral products and phosphopeptide mapping showed that the 100 and 68 kDa proteins are coded by cellular genes. Phosphoproteins were undetectable in control resting lymphocytes. The 68 and the 100 kDa proteins were phosphorylated to different extents in proliferating lymphocytes, either stimulated by the growth factor IL-2, or transformed by M-MuLV (lacking the oncogene coded kinase). In fibroblasts transformed by A-MuLV, phosphotyrosine antibodies identified 2 proteins of 120 and 70 kDa. By immunological cross-reaction and by phosphopeptide mapping, the first was identified as a 120 kDa form of the v-abl coded kinase. The 70 kDa protein is coded by a cellular gene, is not structurally related to the 120 kDa v-abl kinase, and is different from any phosphotyrosine-containing protein detected in A-MuLV-transformed lymphocytes. These data show that, upon v-abl-induced transformation, phosphorylation at tyrosine takes place also on proteins other than the 160 or 120-kDa oncogene products. In lymphocytes and fibroblasts these proteins are different, suggesting that the cascade of events triggered by the v-abl gene in different cell types involves tyrosine phosphorylation of different specific proteins.

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.

It is known that the HTLV-I-transformed cell line MT4 releases chemotactic activity for monocytes spontaneously. The MT4 monocyte chemoattractant was purified to homogeneity and sequencing of 25 amino acids revealed identity with the C-C chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha/LD78). An anti-MIP-1 alpha/LD78 rabbit antiserum substantially inhibited chemotaxis of the MT4 chemoattractant. MT4 cells constitutively expressed MIP-1 alpha/LD78 but not the C-C chemokines MCP-1, RANTES, and MIP-1 beta/Act2 and the C-X-C chemokines IL-8, gro alpha, and gro beta. MT4-derived MIP-1 alpha/LD78 was active on monocytes but was a weak chemoattractant for polymorphonuclear leukocytes. Thus, MIP-1 alpha/LD78 is a major monocyte chemoattractant released by HTLV-I-transformed T cells. Expression of MIP-1 alpha/LD78, a leukocyte chemotactic and myelosuppressive molecule, may play an important role in the manifestations of HTLV-I-related diseases.

11058 Background: Aromatase inhibitors (AIs) are the first choice for older patients (pts) with estrogen-receptor (ER) positive advanced breast cancer (BC). Yet, response is variable and difficult to predict. Some pts exhibit very good long-lasting response whereas others rapidly progress even after initial response. Factors such as tumor subtype (i.e., luminal A vs B) or BMI have been reported to correlate with response but so far no predictive marker is available. The aim of this study was to identify gene variants associated with AIs response in elderly pts with advanced BC. Methods: Genomic DNA from peripheral blood of 55 pts with locally advanced or metastatic breast cancer was analyzed with DMET Plus GeneChip array (Affymetrix, Santa Clara, CA), appropriateness of data was evaluated with DMET Console and statistically relevant variants were assessed with DMET-Analyzer. Median age was 79 years (IQR, 73-86), all patients had ER-positive HER-2 negative BC. Clinico-pathologic factors were evaluated for ...

The Abelson and Moloney murine leukemia virus complex (A-MuLV/M-MuLV) induces rapidly growing thymic lymphomas following direct injection into the thymus of newborn BALB/c and C57BL/6 mice. Southern blot analysis with a v-abl specific probe not only demonstrated that primary tumors are clonal, but also that the pattern of A-MuLV provirus integration is quite stable in primary tumor cells, as well as in their derived cell lines and clones. Most of the cell samples were able to rearrange the immunoglobulin heavy chain genes in culture, whereas in two cases the T cell receptor gamma chain genes also underwent rearrangement. Since the recombination mechanism is operative only in very immature lymphoid cells, these data provide indirect evidence for the lack of differentiation of A-MuLV cell targets in the thymus.

Experiments were carried out to investigate whether the human T-lymphotropic virus type I (HTLV-I), alone or in combination with a chemical mutagen such as mitomycin C (MMC), has the capacity to damage host chromosomes. Cord-blood T lymphocytes (CBL) were infected by co-cultivation with lethally irradiated HTLV-I-producing cells. Infected and immortalized CBL were then studied for frequencies of sister chromatid exchanges (SCE), chromosome breaks and micronuclei. HTLV-I-infected cells had statistically higher baseline SCE, chromosome aberrations and micronucleus values than the uninfected control CBL. While MMC treatment further augmented these values both in control and in infected lymphocytes, the latter did not show dose-related increases, most likely because of the more pronounced MMC-induced delaying effect on cell progression to mitosis. In view of similar previous observations in mouse lymphocytes carrying the Moloney murine leukemia virus, it is suggested that expression of a common retrovirus gene product, such as the pol endonuclease, might be responsible for the cytogenetic abnormalities observed. In addition to the IL-2 autocrine loop, the direct induction of chromosome damage by HTLV-I in target lymphocytes may be related to the pathogenesis of malignancies associated with HTLV-I infection.