The role of the IFN-inducible p204 as growth regulator was investigated by transfecting an expression vector constitutively expressing p204 into several cell lines. Like pRB and p107, p204 is a potent growth inhibitor in sensitive cells, as demonstrated by the cell focus assay. Since stable transfectants of sensitive lines constitutively overexpressing p204 could not be established in vitro, we inserted the 204 cDNA into a vector bearing an heavymetal-inducible promoter. Here we show that proliferation of B6MEF ®broblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G 1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G 1 . Moreover, the role of p204 in the control of cell growth in vivo was investigated by generating transgenic mice in which the I® 204 gene was constitutively expressed in all tissues. To this end, expression vectors bearing the 204 cDNA under the control of the SV40 viral promoter were constructed. The overexpression of the p204 transgene achieved by injecting fertilized mouse eggs with these vectors was compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development. These ®ndings indicate that p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation.

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The occurrence of apoptosis and its contribution to growth-phase transitions in the rat ascites hepatoma Yoshida AH-130 have been evaluated. Apoptosis was observed by light microscopy in both exponentially-growing and stationary tumours as characteristic chromatin condensation and compacting of the cytoplasm, but the frequency of apoptotic bodies (apoptotic index) was four-fold higher in stationary than in logarithmic-growing tumours. Apoptosing cells exhibited strong immunocytochemical reactivity for 'tissue' transglutaminase, and transglutaminase activity was higher in stationary tumour cells. A ladder pattern of DNA fragmentation was revealed by agarose gel electrophoresis which was more pronounced in stationary tumours. The apoptotic bodies were either free or contained in vacuoles within otherwise normal tumour cells, suggesting active engulfment of dead cells by viable homologous cells. Such 'homophagic' disposal of the apoptotic bodies probably accounted for a component of the enhanced cell protein degradation previously observed in stationary AH-130 tumours (Tessitore L, Bonelli G, Cecchini G, Amenta JS, Baccino FM, Arch Biochem Biophys 1987; 255: 372-384). The data indicate that, in concurrence with changes in the cell proliferation rate, modulations of apoptosis play a substantial role in determining the net growth rate of such an anaplastic tumour as the Yoshida AH-130 hepatoma.

The purpose of this study was to characterize the stages in the development of thymidine-induced cell death. L-cells were characterized by both morphologic and quantitative techniques and evaluated at 24, 48, and 72 h of treatment. Cells first enlarged (stage I); about 50% of these enlarged cells then decreased in size with blebbing and compacting (stage II). This residual cell body transformed into a smooth eosinophilic hyaline body (stage III) by 72 h, many of which could be identified within the vacuolar system of viable cells. These changes were reflected in morphologic counts and Coulter sizing. Cell death (loss of labeled DNA) began in stage II and was most prominent in stage III. No cleavage of DNA into oligonucleosomal fragments was detected by agarose gel electrophoresis at any stage. The similarity of these changes to the complete spectrum of apoptosis in vivo is discussed.

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