Franco Tatò

Interaction with normal cells suppresses the transformed phenotype of v-myc-transformed quail muscle cells

We have analyzed mixed cultures of normal mammalian fibroblastic cells and transformed quail myoblasts to investigate whether the presence of an excess of normal cells could suppress the phenotype of transformed quail cells. In such mixed cultures, only v-myc-transformed cells were growth-arrested, whereas v-src-transformed myoblasts were essentially unaffected. Growth arrest appeared to reflect reversion from the transformed state, including re-expression of the myogenic differentiation program. The v-myc-transformed myoblasts were phenotypically corrected also by differentiating normal quail myoblasts, giving rise to hybrid myotubes containing nuclei from both cell types. The differential behavior of transformed cells closely paralleled the efficiency with which they established metabolic cooperation with adjacent normal cells. Our results indicate that unrestrained proliferation associated with transformation is responsible for v-myc-induced block of myogenic differentiation.

Interaction of Retroviral Oncogenes With The Differentiation Program of Myogenic Cells

Publisher Summary This chapter identifies common features in the interaction between viral oncogenes and in vitro differentiating cells by comparing the behavior of retrovirus-infected muscle cells with that of other cell types. The chapter also discusses the role of oncogenes in the relationship between acquisition of the transformed state, subversion of proliferation control, and interference with the expression of a differentiation program. This complex and multifarious phenotype may well represent the in vitro counterpart of the pathologists observations in spontaneous tumors—benign tumors are reasonably well differentiated, whereas malignant ones tend to be anaplastic. Myogenesis certainly provides the best known model for studying the mechanisms governing the transition from a determined to an overtly differentiated state, defined at the molecular level by the activation and expression of cell type-specific gene products and, at the cellular level, by formation of tissue. Transforming retroviruses represent excellent tools for the study of cell transformation in vitro. They carry in their genome specific sequences, known as oncogenes that are responsible for tumor formation in vivo and cell transformation in vitro .

Transcription of muscle-specific genes is repressed by reactivation of pp60v-src in postmitotic quail myotubes.

Quail myogenic cells infected with temperature sensitive (ts) mutants of Rous sarcoma virus (RSV) exhibit a temperature-dependent transformation and block of differentiation. When the cells are allowed to differentiate at the restrictive temperature (41 degrees C) and then shifted back to the permissive temperature (35 degrees C), a sharp reduction in the accumulation of muscle-specific mRNAs is observed, following reactivation of the transforming protein pp60v-src. A kinetic analysis of this down-regulation reveals that the reduction in the accumulation of muscle-specific transcripts occurs fairly rapidly within 6 to 20 h after the shift back, depending on the mRNA analyzed. Studies on transcription of endogenous muscle-specific genes and a transfected chloramphenicol acetyltransferase reporter gene under the control of muscle-specific promoters, at the different temperatures, suggest that the oncogene exerts its control mainly at the transcriptional level. On the contrary, transcription of the CMD1 gene, the avian homolog of the mouse muscle regulatory MyoD gene, is not significantly affected by the oncogene both in proliferating myoblasts and in myotubes shifted back to 35 degrees C. These findings are consistent with the conclusion that v-src blocks myogenesis by controlling transcription of muscle-specific genes independently of cell proliferation. Furthermore, they suggest the existence of an alternative pathway, not requiring the silencing of CMD1 transcription, through which the oncogene exerts its effect.

Adenovirus infection induces reentry into the cell cycle of terminally differentiated skeletal muscle cells.

Different cell types in the body of higher animals undergo terminal differentiation. In such a process, cells acquire specialized functions and irreversibly lose their ability to divide, therefore entering the postmitotic state. Terminally differentiated cells do not proliferate in response to growth factors or following the expression of activated, retroviral oncogenes. In this paper we demonstrate that adenovirus infection is an efficient and convenient means to induce terminally differentiated cells to reenter the cell cycle. These findings constitute a first step toward defining the molecular determinants of the irreversible withdrawal from the cell cycle of terminally differentiated cells. They may also open the way to therapeutic applications.

Role of Gas1 down-regulation in mitogenic stimulation of quiescent NIH3T3 cells by v-Src

Quiescent mammalian fibroblasts can be induced to re-enter the cell cycle by growth factors and oncoproteins. We studied the pathway(s) through which v-Src, the oncogenic tyrosine kinase encoded by the v-src oncogene of Rous sarcoma virus, forces serum-starved NIH3T3 cells to enter S-phase. To this purpose, we isolated and characterized a polyclonal population of NIH3T3 cells transformed by the MR31 retroviral vector, encoding G418 resistance and the v-src temperature-sensitive allele from the mutant ts LA31 PR-A. NIH(MR31) cells displayed a temperature-conditional transformed phenotype and could be made quiescent by serum deprivation at the restrictive temperature. Serum stimulation or thermolabile v-Src reactivation induced entry into S-phase to a comparable extent, although with different kinetics. The data suggest that v-Src mitogenic activity involves early activation of the Erk1/Erk2 MAP kinases with very little tyrosine phosphorylation of the Shc adaptor proteins at least during the early stages of v-Src reactivation and that v-Src-induced S-phase entry was strongly inhibited by drugs affecting MEK or PI 3-kinase. Our results also suggest that down-regulation of gas1 gene expression plays an important role in regulating the efficiency of entry into S-phase triggered by reactivated v-Src and that Gas1 down-regulation does not require PI 3-kinase dependent signals.

Transcriptional down-regulation of myogenin expression is associated with v-ras-induced block of differentiation in unestablished quail muscle cells.

Unestablished quail myoblasts were infected with a retroviral vector encoding the oncogenic form of H-Ras in order to investigate the mechanism by which this oncoprotein interferes with terminal differentiation. Primary quail myogenic cells exhibit the simultaneous expression of the muscle regulatory genes myf-5, MyoD and myogenin in proliferative conditions. v-ras-transformed myoblasts displayed an altered growth control and lost the competence for terminal differentiation. When expression of myogenic regulatory genes was analysed, it was immediately apparent that the difference between normal and v-ras-transformed cells was limited to a severely decreased level of myogenin expression. Forced expression of exogenous myogenin in v-ras-transformed quail myoblasts led to a striking recovery of the competence for terminal differentiation. The present data show that: (i) repression of myogenin expression is linked to the differentiation defective phenotype of quail myoblasts transformed by v-ras as well as other retroviral oncogenes; (ii) correction of the differentiation-defective phenotype of v-ras-transformed myoblasts by exogenous myogenin entailed reactivation of endogenous myogenin and of the E-box-dependent transactivating function. These results strongly indicate that myogenin expression plays a central role in regulating the transition into the terminally differentiated state and that its transcriptional down-regulation represents a nodal step in v-ras-induced block of differentiation.

Selective repression of myoD transcription by v-Myc prevents terminal differentiation of quail embryo myoblasts transformed by the MC29 strain of avian myelocytomatosis virus

We have investigated the mechanism by which expression of the v-myc oncogene interferes with the competence of primary quail myoblasts to undergo terminal differentiation. Previous studies have established that quail myoblasts transformed by myc oncogenes are severely impaired in the accumulation of mRNAs encoding the myogenic transcription factors Myf-5, MyoD and Myogenin. However, the mechanism responsible for such a repression remains largely unknown. Here we present evidence that v-Myc selectively interferes with quail myoD expression at the transcriptional level. Cis-regulatory elements involved in the auto-activation of qmyoD are specifically targeted in this unique example of transrepression by v-Myc, without the apparent participation of Myc-specific E-boxes or InR sequences. Transiently expressed v-Myc efficiently interfered with MyoD-dependent transactivation of the qmyoD regulatory elements, while the myogenin promoter was unaffected. Finally, we show that forced expression of MyoD in v-myc-transformed quail myoblasts restored myogenin expression and promoted extensive terminal differentiation. These data suggest that transcriptional repression of qmyoD is a major and rate-limiting step in the molecular pathway by which v-Myc severely inhibits terminal differentiation in myogenic cells.

Tumor promoters enhance v-myc-induced focus formation in mammalian cell lines.

The proliferation of normal fibroblastic cells in culture is under highly effective control(s) leading to growth arrest a t high cell density or in the absence of anchorage to a solid substratum. In contrast, transformed cells are not subjected to these restrictions and are usually characterized by increased saturation density and anchorageindependent proliferation. The ability of transformed cells to produce discrete focal areas (foci) in a dense monolayer and to form colonies in soft agar has been extremely useful in identifying viral and cellular oncogenes. However, phenotypic consequences of the expression of exogenous oncogenes may vary greatly depending on host-cell characteristics. For example, src, the Rous sarcoma virus (RSV) oncogene, induces transformation in a number of cell types of avian and mammalian origin,’ it causes no detectable effects in chick macrophages? and it induces growth arrest and terminal differentiation in a rat phaeochromocytoma cell line.3 Another example of hostdependent effects is represented by the differential transforming potential of myc, the avian MC29 virus oncogene, in avian and mammalian fibroblasts. Expression of exogenous myc in nonestablished avian fibroblasts appears to confer an easily detectable alteration of growth control resulting in focus formation, anchorage independence and increased growth rate.4 Transfection studies with molecularly cloned v-myc also suggest that this oncogene may induce focal outgrowth of transformed cells in avian,’ but not in mammalian early passage fibroblast^.^* We were interested in investigating the reason(s) behind this differential transforming potential of the myc oncogene. The first aspect of the issue we chose to investigate was the role of adjacent normal cells in modulating the focal outgrowth of cells bearing the myc oncogene. To this end we measured the ability of small numbers of NIH 3T3 carrying different oncogenes to overgrow an excess of surrounding normal