Constitutive c-myc expression enhances proliferation of differentiating F9 teratocarcinoma cells

The c-myc protooncogene is expressed in many tumor cells as well as during normal development. In order to study the role of c-myc in differentiation, proliferation and tumorigenicity of F9 mouse teratocarcinoma cells, the pSVmyc1 plasmid constitutively expressing an active c-myc oncogene was introduced into F9 stem cells by cotransfection with the selectable marker RSVneo. Enhanced expression of c-myc did not alter the properties of F9 stem cells. Prolonged proliferation during retinoic acid induced differentiation was observed in cell clones constitutively expressing c-myc. In contrast, as determined by morphology, by immunocytochemistry for markers specific for stem cells and differentiated derivatives, and by Northern hybridization for mRNAs specific for differentiated cells, differentiation was neither inhibited nor delayed by constitutive c-myc expression. Tumorigenicity of stem cells as well as retinoic acid-treated cells--as measured by soft agar cloning efficiency and tumor formation in syngenic mice--was not altered by SVmyc1. We conclude that in F9 teratocarcinoma cells down-regulation of c-myc is related to arrest of proliferation rather than differentiation.     

c-myc and c-fos expression in differentiating mouse primary keratinocytes.

Expression of the myc and fos genes has been monitored in mouse primary keratinocytes after induction of terminal differentiation by calcium or tetradecanoylphorbol acetate (TPA). myc RNA levels in growing cells are very high and remain elevated even at late times after calcium-induced differentiation. Thus, keratinocytes provide the first example of normal primary cells with persistent c-myc expression irrespective of their proliferative or differentiated state. fos expression is also relatively unaffected by addition of calcium. In contrast to calcium, TPA-induced differentiation is accompanied by dramatic changes in proto-oncogene expression: marked c-fos induction and considerable although transient decrease in c-myc expression. These effects might be important for the keratinocyte response to TPA: TPA treatment of a keratinocyte cell line (RBK) resistant to this substance has no effect on c-myc expression and leads only to minimal c-fos induction. In these cells full fos induction can still be triggered by addition of fresh medium. Thus, the fos gene in normal keratinocytes is inducible through at least two independent mechanisms, only one of which has been lost during derivation of the TPA-resistant cell line.     

Transforming growth factor alpha dramatically enhances oncogene-induced carcinogenesis in transgenic mouse pancreas and liver.

To characterize the effect(s) of transforming growth factor alpha (TGF alpha) during multistage carcinogenesis, we examined tumor development in pancreas and liver of transgenic mice that coexpressed TGF alpha with either viral (simian virus 40 T antigens [TAg]) or cellular (c-myc) oncogenes. In pancreas, TGF alpha itself was not oncogenic, but it nevertheless dramatically accelerated growth of tumors induced by either oncogene alone, thereby reducing the host life span up to 60%. Coexpression of TGF alpha and TAg produced an early synergistic growth response in the entire pancreas together with the more rapid appearance of preneoplastic foci. Coexpression of TGF alpha and c-myc also accelerated tumor growth in situ and produced transplantable acinar cell carcinomas whose rate of growth was TGF alpha dependent. In liver, expression of TGF alpha alone increased the incidence of hepatic cancer in aged mice. However, coexpression of TGF alpha with c-myc or TAg markedly reduced tumor latency and accelerated tumor growth. Significantly, expression of the TGF alpha and myc transgenes in hepatic tumors was induced up to 20-fold relative to expression in surrounding nonneoplastic liver, suggesting that high-level overexpression of these proteins acts as a major stimulus for tumor development. Finally, in both pancreas and liver, combined expression of TGF alpha and c-myc produced tumors with a more malignant (less differentiated) appearance than did expression of c-myc alone, consistent with an influence of TGF alpha upon the morphological character of c-myc-induced tumor progression. These findings demonstrate the importance of TGF alpha expression during multistage carcinogenesis in vivo and point to a major role for this growth factor as a potent stimulator of tumor growth.     

Effects of growth stimulatory factors on mitogenicity and c-myc expression in poorly differentiated and well differentiated human colon carcinoma cells

We demonstrate the differential sensitivity of poorly differentiated and well differentiated human colon carcinoma cells to nutrients alone or to nutrients and polypeptide growth factors under completely serum-free conditions. 3H-Thymidine incorporation into trichloroacetic acid precipitable material and autoradiographic analysis indicated that nutrient replenishment alone was sufficient to initiate DNA synthesis in quiescent poorly differentiated cells, whereas defined polypeptide growth factors produced no additional effect. In contrast, well differentiated cells were mitogenically stimulated to a much greater extent by growth factors (epidermal growth factor + insulin + transferrin), than by nutrient replenishment alone. Expression of the c-myc protooncogene was increased approximately 5-fold after growth factor addition to the well differentiated cells. Maximal expression of c-myc occurred at 4 h post stimulation. In contrast, nutrients resulted in only a slight up-regulation of c-myc (1.8-fold) at approximately 90 min after addition. Addition of nutrients and/or growth factors to the poorly differentiated colon carcinoma cells resulted in an initial decline in c-myc expression (90 min), presumably due to removal of endogenous growth stimulators. Expression of c-myc returned to baseline levels by 24 h after additions. The results indicate that differential sensitivity to polypeptide growth factors is related to differentiation status in this model system and suggest that the insensitivity of poorly differentiated cells to exogenous growth factors may be due to a greater production of autocrine growth stimulators.     

Comparative study of tumor angiogenesis and immunohistochemistry for p53, c-ErbB2, c-myc and EGFr as prognostic factors in gastric cancer

Gastric cancer (GC) continues to be a highly aggressive malignancy with poor prognosis and low survival rates. The survival of patients with GC depends mainly on the stage of the disease, with early GC having a 5 year survival of 90-100% and advanced tumors a 5 year survival of 15-25%. The role of other prognostic factors in these tumors is still under investigation. 28 gastric dysplasia, 45 Early GC and 98 Advanced Gastric Cancers were evaluated for expression of the oncogenes p53, c-ErbB2, c-myc and the EGFr in paraffin-embedded material utilizing Avidin-Biotin immunohistochemistry techniques. In 34 cases of GC microvessel density (MVD) was determined in CD34 stained sections. Statistical correlations with stage, histologic type, differentiation degree, location, size, ploidy patterns and overall survival were done. The Mantel-Cox test was performed to evaluate which factors had an independent prognostic value. Both, tumor angiogenesis and p53 protein expression were statistically associated (95% confidence intervals) with overall survival in patients with GC. p53 protein expression was also correlated with cardial location, nodal involvement and tumor stage. c-ErbB2 may recognize a group of highly aggressive well differentiated adenocarcinomas with worse prognosis. c-myc was also significantly enhanced in well differentiated tumors. EGFr showed no significant associations. Mantel-Cox was performed to compare the prognostic value of tumor stage, p53 protein expression and tumor angiogenesis. Tumor angiogenesis was the most important prognostic indicator to predict overall survival in our series. p53 expression was not independent and did not provide additional prognostic information to tumor stage. Our study suggests that angiogenesis as demonstrated by microvessel counts in CD34 stained sections is a significantly important prognostic factor for predicting survival in gastric cancer.