Increased activity of c-Src and Csk in fibroblasts transformed by v-src oncogene.

When c-Src and v-Src were immunoprecipitated together from hamster fibroblasts transformed by Rous sarcoma virus containing v-src oncogene, the total Src activity was almost threefold higher compared to c-Src activity in the control cells. The activity of v-Src immunoprecipitated separately, however, accounting for only 40% of the total Src activity, indicating that c-Src is activated upon transformation. An increased activity of Csk was also found in RSV-transformed cells. It decreased upon serum stimulation in parallel with an increase in Src kinase activity. In nontransformed cells, serum stimulation induced an enhanced Csk activity, but no changes in c-Src activity were observed. This may suggest that Csk may have more functions in hamster fibroblasts, in addition to its inhibitory effect on c-Src.     

Hormonal regulation of expression of the oncogene v-src in mouse fibroblasts NIH 3T3

NIH 3T3 cells were transfected by plasmid containing v-src under control of hormone-regulated LTR MMTV (pMLsrc10). This plasmid caused the foci of morphologically transformed cells. The transformed cells induced rapidly growing tumours in nude mice. In the presence of dexamethasone the efficiency of NIH 3T3 cell transformation increased ten times, while tumourigenicity remained unchanged.     

Long-range effects of direct-hit ultraviolet and particle radiation in oncogene activation

A simple statistical analysis shows that the oncogene-activation effect of chemical carcinogens cannot be explained if one takes into account only short-range effects. As one of the most probable solid state physical long-range effects, the generation at the site of carcinogen binding of travelling solitary waves, which can interfere with DNA-blocking protein interactions, is discussed. It has been shown that the direct hit carcinogenic effects on DNA by ultraviolet--or particle radiation can also be explained by the generation of solitary waves (in the latter case the first step is a collective plasma oscillation which decays to individual local excitations and ionizations).     

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.     

Effects of transformation with the v-src oncogene on inositol phosphate metabolism in rat-1 fibroblasts. D-myo-inositol 1,4,5,6-tetrakisphosphate is increased in v-src-transformed rat-1 fibroblasts and can be synthesized from D-myo-inositol 1,3,4-trisphosphate in cytosolic extracts

Rat-1 fibroblasts transformed with the v-src oncogene show a 6-fold increase in the apparent amount of an inositol polyphosphate which has a high performance liquid chromatography (HPLC) elution characteristic of the D/L-myo-inositol 1,4,5,6-tetrakisphosphate enantiomeric pair (Johnson, R.M., Wasilenko, W.J., Mattingly, R.R., Weber, M.J., and Garrison, J.C. (1989) Science 246, 121-124). By chemical and enzymatic analysis, the structure of this compound produced in both normal and v-src-transformed rat-1 fibroblasts has been determined to be principally D-myoinositol 1,4,5,6-tetrakisphosphate (D-Ins(1,4,5,6)P4). Chronic stimulation with endothelin-1 in the presence of Li+ significantly increased the amount of D/L-Ins(1,4,5,6)P4 only in the v-src-transformed rat-1 cells, suggesting that production of this compound may be remotely coupled to long term agonist-induced phosphatidylinositol turnover. Further evidence for such a link is provided by the progressive loss of D-Ins(1,4,5,6)P4 from the normal cells deprived of serum stimulation. To define a possible synthetic pathway for D-Ins(1,4,5,6)P4, cytosolic extracts of normal and v-src-transformed cells were incubated with [3H]inositol polyphosphates, and the reaction products were identified by HPLC elution and chemical analysis. Although inositol 1,3,4-trisphosphate 6-kinase activity was prominent in extracts of both normal and transformed cells, only the cytosol from v-src-transformed cells ultimately formed measurable amounts of D-Ins(1,4,5,6)P4 from [3H]inositol 1,3,4-trisphosphate. Approximately 6% of 0.1 microM inositol 1,3,4-trisphosphate was converted to D-Ins(1,4,5,6)P4 during a 2-h incubation at 37 degrees C. Inositol pentakisphosphate was identified as a likely intermediate in this conversion, and extracts of both normal and transformed cells converted [3H]inositol 1,3,4,5,6-pentakisphosphate to D-Ins(1,4,5,6)P4. The synthetic pathway described is consistent with the long term regulation of D/L-Ins(1,4,5,6)P4 levels in rat-1 fibroblasts seen in response to src transformation, serum withdrawal, and chronic endothelin treatment, and identifies several new potential interactions between the pathways of inositol polyphosphate metabolism and those of src transformation.     

Expression of the v-src or v-fps oncogene increases fructose 2,6-bisphosphate in chick-embryo fibroblasts. Novel mechanism for the stimulation of glycolysis by retroviruses.

The concentration of fructose 2,6-bisphosphate and the activity of 6-phosphofructo-2-kinase are increased after infection of chick-embryo fibroblasts with the Rous sarcoma virus, or with a temperature-sensitive mutant of this virus at the permissive, but not at the non-permissive, temperature. This is observed after transformation by retroviruses carrying either the v-src or v-fps, but not the v-mil and/or v-myc, oncogenes. Comparison of the effects of the Rous sarcoma virus with those of phorbol myristate acetate on fructose 2,6-bisphosphate suggests that both result from the stimulation of a step which is rate-limiting for 6-phosphofructo-2-kinase activation and which is also controlled by protein kinase C.     

Secreted proteins of normal andmyc-ras oncogene transformed rat embryo fibroblasts

Quiescent cultures of rat embryo fibroblasts synthesize and secrete several proteins in response to mitogenic stimulation. Two of these proteins have been characterized in this study and the effect of oncogenic transformation on these proteins was monitored. A serum induced 48,000 protein was shown to be related to plasminogen activator inhibitor while another serum-induced protein ofM _r 45,000 was found to be an inhibitor of DNA synthesis. Transformation of rat embryo fibroblasts with oncogenesmyc andras resulted in drastic reduction in the level of these proteins. The reduced levels of protease inhibitor may be responsible for the loss of anchorage dependence of the transformed cells. The DNA synthesis inhibitor protein may act as a negative growth regulator and reduced levels of this protein inmyc-ras transformed cells may accelerate the proliferation of these cells.     

v-src transformation of rat embryo fibroblasts. Inefficient conversion to anchorage-independent growth involves heterogeneity of primary cultures

To clarify whether a single oncogene can transform primary cells in culture, we compared the transforming effect of a recombinant retrovirus (ZSV) containing the v-src gene in rat embryo fibroblasts (REFs) to that in the rat cell line 3Y1. In the focus assay, REFs exhibited resistance to transformation as only six foci were observed in the primary cultures as opposed to 98 in 3Y1 cells. After G418 selection, efficiency of transformation was again somewhat lower with REFs compared to that with 3Y1 cells, but the number of G418-resistant REF colonies was much greater than the number of foci in REF cultures. Furthermore, while 98% of G418-resistant colonies of ZSV-infected REFs were morphologically transformed, only 25% were converted to anchorage- independent growth, as opposed to 100% conversion seen in ZSV-infected 3Y1 cells. The poor susceptibility of REFs to anchorage-independent transformation did not involve differences in expression and subcellular distribution of p60v-src, or its kinase activity in vitro and in vivo. It rather reflected a property of the primary cultures, as cloning of REFs before ZSV infection demonstrated that only 2 out of 6 REF clones tested were permissive for anchorage-independent growth. The nonpermissive phenotype was dominant over the permissive one in somatic hybrid cells, and associated with organized actin filament bundles and a lower growth rate, both before and after ZSV infection. These results indicate that the poor susceptibility of REFs to anchorage-independent transformation by p60v-src reflects the heterogeneity of the primary cultures. REFs can be morphologically transformed by p60v-src with high efficiency but only a small fraction is convertible to anchorage- independent growth. REF resistance seems to involve the presence of a suppressor factor which may emerge from REF differentiation during embryonic development.     

Neoplastic transformation of normal and carcinogen-induced preneoplastic Syrian hamster embryo cells by the v-src oncogene.

The ability of cloned Rous sarcoma virus (RSV) DNA encoding the v-src oncogene to neoplastically transform normal, diploid Syrian hamster embryo (SHE) cells was examined. Transfection of RSV DNA into early passage SHE cells resulted in a low but significant number of tumors when treated cells were injected into nude mice. Tumors formed with a low frequency (two tumors out of ten sites injected) and only after a long latency period (14 weeks). In contrast to the normal SHE cells, several different carcinogen-induced preneoplastic immortal SHE cell lines were highly susceptible to transformation by the v-src oncogene to the neoplastic phenotype. Tumors formed with high efficiency and a short latency period (less than 3 weeks). Further studies were performed to determine the basis for the inefficient transformation of the normal SHE cells. NeoR clones isolated after cotransfection of SHE cells with pSV2-neo and RSV DNAs were neither morphologically altered nor immortal and did not contain detectable levels of the v-src gene product. These results suggest that neoplastic transformation by v-src DNA in the normal cells is initially suppressed. However, cells from a v-src-induced tumor expressed v-src RNA, and antibody to v-src protein precipitated from the tumor cells a 60,000-molecular-weight protein which displayed protein kinase activity. Karyotypic analyses confirmed that the tumor was derived from Syrian hamster cells and suggested that it was clonal in nature. These results indicate that the v-src oncogene was primarily responsible for neoplastic transformation of SHE cells. In contrast to the results with the v-src oncogene, our previous studies showed that v-Ha-ras oncogene alone is unable to induce neoplastic transformation of SHE cells. Furthermore, the v-myc oncogene was able to compliment v-Ha-ras to neoplastically transform SHE cells, while cotransfection with v-src plus v-myc did not increase the incidence of tumors.     

Analysis of p60v-src mutants carrying lesions involved in temperature sensitivity.

Analysis of the src genes of three temperature-sensitive (ts) mutants of Rous sarcoma virus (tsNY68, tsNY72-4, and PA104) showed that each has two C-terminal mutations in the kinase domain required for temperature sensitivity, as assayed by morphological alteration and anchorage-independent growth. In all three mutants, one of the mutations is a valine-to-methionine change at position 461. To assess the contribution of each mutation to the biochemical properties of the src protein, we analyzed the kinase activity and the interaction with cellular proteins p50 and p90 of recombinant src gene products in which only one mutation was combined with wild-type src sequences. Chimeric src protein containing only the Met-461 mutation was indistinguishable from the wild type by all criteria examined, while the effect of the second C-terminal mutation alone varied with the defectiveness of the parental ts mutant. The second mutation alone, while not sufficient to cause ts transformation, altered p60src complex formation with cellular proteins p50 and p90 and altered the in vitro thermolability of src kinase activity. The results indicate that these biochemical properties of p60src are more sensitive to mutation than others, such as in vivo kinase activity, which require more profound structural alterations.     

Ultrastructural effects of colchicine,vinblastine, and taxol in drug-sensitive and multidrug-resistant J 774.2 cells

Summary Cell lines derived from the murine macrophage-like cell J 774.2 are resistant to the cytotoxic effects of colchicine, vinblastine, and taxol. These multidrug-resistant (MDR) cells overproduce a family of 130–150 kDa P-glycoproteins (P-gp) associated with the plasma membrane region and display other typical features of the MDR phenotype. Ultrastructural analysis of drug-treated cells indicated that although hallmark structural effects engendered by each drug at efficacious doses were profound in the drug-sensitive J 774.2 cells, they were not evident in the similarly treated MDR cell lines. Thus, MDR phenotypic expression involved maintaining drug levels at subthreshold values so as to preclude the advent of these morphologic changes, and allowed vital tubulin-associated cellular processes, including replication, to occur. Using a polyclonal antibody specific for the P-gp, electron microscopic immunocytochemical evidence is presented for substantial association of P-gp with the plasma membrane/cell surface in the resistant cells which was not demonstrable in the drug-sensitive J 774.2 cells. This key cell surface localization of P-gp is germane to the postulated transport and related mechanisms whereby P-gp may play a pivotal role in endowing cells with multidrug resistance.     

Effects of bradykinin on cell volume and intracellular pH in NIH 3T3 fibroblasts expressing the ras oncogene.

BCECF fluorescence has been applied to determine intracellular pH (pHi) in NIH 3T3 fibroblasts expressing the Ha-ras oncogene (+ras) and otherwise identical cells not expressing the oncogene (-ras). In +ras cells, pHi is significantly more alkaline (6.79 +/- 0.03 n = 12) than in -ras cells (6.64 +/- 0.02, n = 8). Bradykinin (100 nmol/l) leads to intracellular alkalinization in both +ras (to 6.96 +/- 0.04, n = 12) and -ras cells (to 6.85 +/- 0.02, n = 8). The effect of bradykinin is completely abolished in the presence of dimethylamiloride (100 mumol/l), which does not modify pHi in the absence of bradykinin. Similar to bradykinin, cell shrinkage by addition of 15 mmol/l NaCl to the extracellular fluid leads to intracellular alkalinization (by 0.08 +/- 0.01, n = 15). Cell volume is significantly greater in +ras cells (2.7 +/- 0.4 pl, n = 15) than in -ras cells (2.2 +/- 0.4 pl, n = 15). Bradykinin leads to cell shrinkage in both +ras cells (by 7 +/- 1%, n = 17) and -ras cells (by 5 +/- 1%, n = 15). The effect of bradykinin on cell volume can be reversed by the reduction of extracellular NaCl concentration by 15 mmol/l NaCl in +ras cells and by 7 mmol/l NaCl in -ras cells. This maneuver completely abolishes (in -ras cells) or blunts (in +ras cells) the alkalinizing effect of bradykinin. In conclusion, +ras cells are more alkaline than -ras cells. Bradykinin leads to further intracellular alkalinization by activation of the Na+/H(+)-exchanger, at least in part secondary to hormone-induced cell shrinkage.     

Early effects of PP60v-src kinase activation on caveolae

Members of the nonreceptor tyrosine kinase family appear to be targeted to caveolae membrane. We have used a Rat-1 cell expressing a temperature sensitive pp60^v-src kinase to assess the initial changes that take place in caveolae after kinase activation. Within 24–48 h after cells were shifted to the permissive temperature, a set of caveolae-specific proteins became phosphorylated on tyrosine. During this period there was a decline in the caveolae marker protein, caveolin-1, a loss of invaginated caveolae, and a 70% decline in the sphingomyelin content of the cell. One of the phosphorylated proteins was caveolin-1 but it was associated in coimmunoprecipitation assays with both a 30 kDa and a 27 kDa tyrosine-phosphorylated protein. Finally, the cells changed from having a typical fibroblast morphology to a rounded shape lacking polarity. In light of the recent evidence that diverse signaling events originate from caveolae, pp60^v-src kinase appears to cause global changes to this membrane domain that might directly contribute to the transformed phenotype. J. Cell. Biochem. 71:524–535, 1998. © 1998 Wiley-Liss, Inc.     

Effects of anesthesia-induced modest hypothermia on cellular radiation sensitivity

To assess the mechanisms of modest hypothermia (MH) and its effects on cellular radiation response, a model of anesthesia-induced modest hypothermia (AIMH) in the adult mice and a model of pure MH in the newborn mice were established. The survival rate of lethally irradiated mice was increased to 72% through AIMH before irradiation. Both apoptosis and necrosis of human fetal bone marrow CD34+ hematopoietic stem cells cultured under MH were significantly decreased as detected by MTT and flow cytometry, with three-color labeled by PE-CD34+/ FITC-AnnexinV /7AAD. The survival and proliferation of mouse bone marrow MNC treated with MH after irradiation were also increased. The MH exerted similar protective effects on the leukemia cell lines A20, HL60, K562 to the normal bone marrow cells, but it enhanced the radiation sensitivity of leukemia cell line FBL3 and mouse melanoma B16F10. No effects have been found on the radiation sensitivity of those cells treated with MH before irradiation. The results also show     

Lack of sensitivity of primary Fanconi's anemia fibroblasts to UV and ionizing radiation

Clinical observations and theoretical considerations suggest some degree of radiosensitivity in Fanconi's anemia (FA), but experimental evidence remains controversial. We tested the sensitivity of primary skin fibroblast cultures from all known FA complementation groups to ionizing radiation and ultraviolet light using conventional cell growth and colony formation assays. In contrast to previous studies, and because FA fibroblasts grow and clone poorly at ambient oxygen, we performed our sensitivity tests under hypoxic cell culture conditions. Fibroblast strains from healthy donors served as negative controls and those from patients with ataxia telangiectasia (AT) and Cockayne syndrome (CS) as positive controls. We observed interstrain variation but no systematic difference in the response of FA and non-FA control fibroblasts to ionizing radiation. After exposure to UV radiation, only complementation group A, G and D2 strains displayed values for colony formation EC50 that were intermediate between those for the negative and positive controls. Because of considerable interstrain variation, minor alterations of the response of individual FA strains to ionizing and UV radiation should be interpreted with caution and should not be taken as evidence for genotype-specific sensitivities of primary FA fibroblasts. All together, our data indicate neither systematic nor major sensitivities of primary FA fibroblast cultures of any complementation group grown under hypoxic cell culture conditions to ionizing or UV radiation.