Suppression of the progression phenotype in somatic cell hybrids occurs in the absence of altered adenovirus type 5 gene expression.

Abstract

In the present study we have analyzed the genetic regulation of increased expression of transformationassociated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (Ell-NMT'") and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFCIP). Parental Ell-NMT"e°cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFP cells exhibited phenotypes opposite to those of E11-NMT'e°cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFCaP and Ell-NMTn" cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which stil exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with Ell-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 ElA and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in AdS-transformed cells and that this suppression is not associated with changes in the expression of AdS transforming genes.Although recent advances have led to a better understanding of the potential role of defined genetic elements in the etiology of cancer, the molecular details by which a cell becomes transformed and ultimately evolves into a population of cells possessing tumorigenic and metastatic potential have not been delineated (reviewed in references 9, 24, and 27). Transforming genes (oncogenes) have been isolated and characterized from viral and mammalian tumor cell genomes which when transferred and expressed in appropriate recipient cells display a dominant-acting phenotype (reviewed in references 2 and 4). In addition to these dominant-acting transforming genetic elements in the genomes of certain tumor cells, several lines of experimental evidence indicate that specific genes exist in normal eucaryotic cells which may function as inhibitors of expression of the transformed phenotype and tumor formation, and these have been referred to as suppressor genes, repressor genes, antioncogenes, or emerogenes (1,3,18,23,28,36). Evidence supporting the existence of suppressor genes has come from (i) the a...

Cooperation between AP1 and PEA3 sites within the progression elevated gene-3 (PEG-3) promoter regulate basal and differential expression of PEG-3 during progression of the oncogenic phenotype in transformed rat embryo cells

“…Similarly, somatic cell hybrids formed between E11-NMT and CREF cells, that display a¯at morphology such as F1 and F2, also fail to form tumors in nude mice (Duigou et al, 1990), although they grow with a low e ciency in agar similar to E11 cells ( Figure 1a). In contrast, speci®c E11-NMT6CREF somatic cell hybrids, that display a round morphology such as R1 and R2, grow with high e ciency in agar, even exceeding that of E11-NMT ( Figure 1a) and they rapidly form tumors in nude mice (Duigou et al, 1990). As observed with E11 cells, the levels of PEG-3 mRNA and protein are reduced in F1 and F2 cells, whereas R1 and R2 display elevated expression of PEG-3 akin to that of E11-NMT and E11-ras R12 cells (Figure 1b,c).…”

“…CREF cells are immortal rat embryo cells that do not form colonies when grown in agar and are devoid of tumorigenic potential when inoculated subcutaneously into athymic nude mice (Fisher et al, 1982;Duigou et al, 1990) (Figure 1a). Similarly, somatic cell hybrids formed between E11-NMT and CREF cells, that display a¯at morphology such as F1 and F2, also fail to form tumors in nude mice (Duigou et al, 1990), although they grow with a low e ciency in agar similar to E11 cells ( Figure 1a).…”

“…CREF cells are immortal rat embryo cells that do not form colonies when grown in agar and are devoid of tumorigenic potential when inoculated subcutaneously into athymic nude mice (Fisher et al, 1982;Duigou et al, 1990) (Figure 1a). Similarly, somatic cell hybrids formed between E11-NMT and CREF cells, that display a¯at morphology such as F1 and F2, also fail to form tumors in nude mice (Duigou et al, 1990), although they grow with a low e ciency in agar similar to E11 cells ( Figure 1a). In contrast, speci®c E11-NMT6CREF somatic cell hybrids, that display a round morphology such as R1 and R2, grow with high e ciency in agar, even exceeding that of E11-NMT ( Figure 1a) and they rapidly form tumors in nude mice (Duigou et al, 1990).…”

“…A useful model for de®ning the genetic and biochemical changes mediating tumor progression is the Ad5/early passage RE cell culture system (Fisher, 1984;Babiss et al, 1985;Duigou et al, 1989Duigou et al, , 1990Duigou et al, , 1991Fisher et al, 1979a,b,c;Reddy et al, 1993;Su et al, 1994Su et al, , 1997Kang et al, 1998a). Transformation of secondary rat embryo (RE) cells by Ad5 is often a sequential process resulting in the acquisition of and further elaboration of speci®c phenotypes by the transformed cell (Fisher et al, 1979a,b,c;Babiss et al, 1985).…”

“…However, a single-treatment with the demethylating agent 5-azacytidine (AZA) results in a stable reversion in transformation progression in 495% of cellular clones (Fisher, 1984;Babiss et al, 1985;Duigou et al, 1989;Reddy et al, 1993;Su et al, 1994). The progression phenotype is also suppressed in somatic cell hybrids formed between normal or un-progressed transformed cells and progressed cells (Duigou et al, 1990(Duigou et al, , 1991Reddy et al, 1993). These ®ndings suggest that progression may result from the activation of speci®c progression-promoting (progression elevated) genes or the selective inhibition of progression-suppressing (progression suppressed) genes, or possibly a combination of both processes (Fisher, 1984;Babiss et al, 1985;Su et al, 1997;Kang et al, 1998a).…”