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Subtraction hybridization applied to terminally differentiating human melanoma cells identified mda-7/IL-24, a cytokine belonging to the IL-10 gene superfamily. Adenoviral-mediated delivery of mda-7/IL-24 (Ad.mda-7) provokes apoptosis selectively in a wide spectrum of cancers in vitro in cell culture, in vivo in human tumor xenograft animal models and in patients with advanced carcinomas and melanomas. In human prostate cancer cells, a role for mitochondrial dysfunction and induction of reactive oxygen species in the apoptotic process has been established. Ectopic overexpression of bcl-xL and bcl-2 prevents these changes including apoptosis induction in prostate tumor cells by Ad.mda-7. We now document that this resistance to apoptosis can be reversed by treating bcl-2 family overexpressing prostate tumor cells with ionizing radiation in combination with Ad.mda-7 or purified GST-MDA-7 protein. Additionally, radiation augments apoptosis induction by mda-7/IL-24 in parental and neomycin-resistant prostate tumor cells. Radiosensitization to mda-7/IL-24 is dependent on JNK signaling, as treatment with the JNK 1/2/3 inhibitor SP600125 abolishes this effect. Considering that elevated expression of bcl-xL and bcl-2 are frequent events in prostate cancer development and progression, the present studies support the use of ionizing radiation in combination with mda-7/IL-24 as a means of augmenting the therapeutic benefit of this gene in prostate cancer, particularly in the context of tumors displaying resistance to radiation therapy owing to bcl-2 family member overexpression.Oncogene (2006) 25, 2339–2348. doi:10.1038/sj.onc.1209271; published online 5 December 2005 [ABSTRACT FROM AUTHOR]

Myocardin (MYOCD) promotes Smad3-mediated transforming growth factor-β (TGF-β) signaling in mouse fibroblast cells. Our previous studies show that TGF-β/SMADs signaling activation enhances epithelial-mesenchymal transition (EMT) in human non-small cell lung cancer (NSCLC) cells. However, whether and how MYOCD contributes to TGF-β-induced EMT of NSCLC cells are poorly elucidated. Here, we found that TGF-β-induced EMT was accompanied by increased MYOCD expression. Interestingly, MYOCD overexpression augmented EMT and invasion of NSCLC cells induced by TGF-β, whereas knockdown of MYOCD expression attenuated these effects. Overexpression and knockdown of MYOCD resulted in the upregulation and downregulation of TGF-β-induced Snail mRNA, respectively. Moreover, MYOCD overexpression promoted TGF-β-stimulated NSCLC cell metastasis in vivo. MYOCD was highly expressed and positively correlated with Snail in metastatic NSCLC tissues. Mechanistically, MYOCD directly interacted with SMAD3 and sustained the formation of TGF-β-induced nuclear SMAD3/SMAD4 complex, facilitating TGF-β/SMAD3-induced transactivation of Snail. Importantly, MYOCD was transcriptionally activated by TGF-β-induced SMAD3/SMAD4 complex and CRISPR/Cas9-mediated silencing of SMAD3/SMAD4 led to a reduction in MYOCD mRNA expression. Taken together, our findings indicate that MYOCD promotes TGF-β-induced EMT and metastasis of NSCLC and identify a positive feedback loop between MYOCD and SMAD3/SMAD4 driving TGF-β-induced EMT.

TROY is a component of the Nogo receptor complex and plays the key role in neuronal survival, migration, and differentiation. Here, we show the up-regulation of TROY in human glioma tissues and cells. Inhibition of TROY expression slowed glioma development in vivo and in vitro. Raf kinase inhibitor (RKIP) was found to interact with TROY. The physical interaction of TROY/RKIP was confirmed via co-immunoprecipitation (co-IP) assays. Furthermore, we found that the TROY/RKIP interaction was enhanced by fetal bovine serum (FBS) exposure, and TROY knockdown also led to down-regulation of NF-κB. Finally, disruption of the TROY/RKIP interaction using the TAT-TROY (234-371 aa) protein reduced the glioma development in xenografted mice. This suggests the TROY/RKIP interaction is a potential target for therapy of gliomas.

Melanoma differentiation associated gene-7 (mda-7) was cloned using subtraction hybridization from terminally differentiated human melanoma cells. Based on structural and functional properties, mda-7 is now recognized as interleukin-24 (IL-24), a new member of the expanding IL-10 gene family. Unique properties of mda-7/IL-24 include its ability to selectively induce growth suppression, apoptosis and radiosensitization in diverse human cancer cells, without causing similar effects in normal cells. The utility of mda-7/IL-24, administered by means of a replication-incompetent adenovirus, as a gene therapy for cancer has recently received validation in patients, highlighting an important phenomenon initially observed in pancreatic tumor cells, namely a 'potent bystander apoptosis-inducing effect' in adjacent tumor cells not initially receiving this gene product. We presently investigated the contribution of mda-7/IL-24 secreted by normal cells in mediating this 'bystander effect', and document that normal cells induced to produce mda-7/IL-24 following infection with recombinant adenoviruses expressing this cytokine secrete mda-7/IL-24, which modifies the anchorage-independent growth, invasiveness, survival and sensitivity to radiation of cancer cells that contain functional IL-20/IL-22 receptors, but not in cancer cells that lack a complete set of receptors. Moreover, the combination of secreted mda-7/IL-24 and radiation engenders a 'bystander antitumor effect' not only in inherently mda-7/IL-24 or radiation-sensitive cancer cells, but also in tumor cells overexpressing the antiapoptotic proteins bcl-2 or bcl-x(L) and displaying resistance to either treatment alone. The present studies provide definitive evidence that secreted mda-7/IL-24 from normal cells can induce direct antitumor and radiation-enhancing effects that are dependent on the presence of canonical receptors for this cytokine on tumor cells. Moreover, we now describe a novel means of enhancing mda-7/IL-24's therapeutic potential by targeting normal cells to produce and release this cancer-specific apoptosis-inducing cytokine, a strategy that could be employed as an innovative way of using this unique gene product for treating metastatic disease.

Abnormalities in cellular differentiation are frequent occurrences in human cancers. Treatment of human melanoma cells with recombinant fibroblast interferon (IFN-beta) and the protein kinase C activator mezerein (MEZ) results in an irreversible loss in growth potential, suppression of tumorigenic properties and induction of terminal cell differentiation. Subtraction hybridization identified melanoma differentiation associated gene-7 (mda-7), as a gene induced during these physiological changes in human melanoma cells. Ectopic expression of mda-7 by means of a replication defective adenovirus results in growth suppression and induction of apoptosis in a broad spectrum of additional cancers, including melanoma, glioblastoma multiforme, osteosarcoma and carcinomas of the breast, cervix, colon, lung, nasopharynx and prostate. In contrast, no apparent harmful effects occur when mda-7 is expressed in normal epithelial or fibroblast cells. Human clones of mda-7 were isolated and its organization resolved in terms of intron/exon structure and chromosomal localization. Hu-mda-7 encompasses seven exons and six introns and encodes a protein with a predicted size of 23.8 kDa, consisting of 206 amino acids. Hu-mda-7 mRNA is stably expressed in the thymus, spleen and peripheral blood leukocytes. De novo mda-7 mRNA expression is also detected in human melanocytes and expression is inducible in cells of melanocyte/melanoma lineage and in certain normal and cancer cell types following treatment with a combination of IFN-beta plus MEZ. Mda-7 expression is also induced during megakaryocyte differentiation induced in human hematopoietic cells by treatment with TPA (12-O-tetradecanoyl phorbol-13-acetate). In contrast, de novo expression of mda-7 is not detected nor is it inducible by IFN-beta+MEZ in a spectrum of additional normal and cancer cells. No correlation was observed between induction of mda-7 mRNA expression and growth suppression following treatment with IFN-beta+MEZ and induction of endogenous mda-7 mRNA by combination treatment did not result in significant intracellular MDA-7 protein. Radiation hybrid mapping assigned the mda-7 gene to human chromosome 1q, at 1q 32.2 to 1q41, an area containing a cluster of genes associated with the IL-10 family of cytokines. Mda-7 represents a differentiation, growth and apoptosis associated gene with potential utility for the gene-based therapy of diverse human cancers.

We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1 transcription factor signaling by dominant negative c-Jun (TAM67) also significantly reduced basal, and to a lesser extent radiation-induced, VEGF promoter function in RT2 cells. Collectively, our data demonstrate that radiation-induced MAPK signaling can both protect cells from radiation-induced cell death as well as enhance protein levels of pro-angiogenic factors such as VEGF. Enhanced VEGF expression in RT2 cells may be mediated via MAPK and JNK pathway signaling which converges upon the AP-1 transcription factor complex.

Cancer is a progressive disease in which a tumor cell temporally develops qualitatively new transformation related phenotypes or a further elaboration of existing transformation associated properties. Subtraction hybridization identified a novel gene associated with transformation progression in mutant adenovirus type 5, H5ts125, transformed rat embryo cells, progression elevated gene-3 (PEG-3). To define the mechanism by which expression of PEG-3 is enhanced as a function of cancer progression a 5'-flanking promoter region of approximately 2.0-kb, PEG-Prom, was isolated, cloned and characterized. The full-length and various mutated regions of the PEG-Prom were linked to a luciferase reporter construct and evaluated for promoter activity during cancer progression. These assays demonstrate a requirement for AP1 and PEA3 sites adjacent to the TATA box region of PEG-3 in mediating basal promoter activity and the enhanced expression of PEG-3 in progressed H5ts125-transformed rat embryo cells. An involvement of AP1 and PEA3 in PEG-3 regulation was also confirmed by electrophoretic mobility shift assays (EMSA) and transfection studies with cJun and PEA3 expression vectors. Our findings document the importance of both AP1 and PEA3 transcription factors in mediating basal and elevated expression of PEG-3 in H5ts125-transformed rat embryo cells displaying an aggressive and progressed cancer phenotype.

Cultured human melanoma cells lose proliferative capacity and terminally differentiate after treatment with the combination of recombinant human fibroblast interferon (IFN-beta) and mezerein (MEZ). Subtraction hybridization of cDNA libraries prepared from actively proliferating human H0-1 melanoma cells from cDNA libraries produced from H0-1 cells treated with IFN-beta + MEZ identifies a novel melanoma differentiation-associated (mda) cDNA, mda-7, that displays elevated expression in differentiation inducer-treated H0-1 cells. mda-7 encodes a novel protein of 206 amino acids with a predicted size of 23.8 kDa. The level of mda-7 mRNA is elevated in actively proliferating normal human melanocytes versus primary and metastatic human melanomas. In the Matrigel-assisted melanoma progression model, mda-7 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. Treatment of human melanomas with IFN-beta + MEZ, and to a lesser extent with MEZ, results in growth suppression and induced or enhanced mda-7 expression. Immunoprecipitation analyses using peptide-derived rabbit polyclonal antibodies detect increases in mda-7 protein, and a higher molecular weight protein of approximately 90 to 100 kDa, in MEZ and IFN-beta + MEZ treated H0-1 cells. mda-7 is a highly conserved gene with an homologous sequence in the genome of yeast. Transfection of mda-7 expression constructs into H0-1 and C8161 human melanoma cells reduces growth and inhibits colony formation. These results confirm that mda-7 has antiproliferative properties in human melanoma cells and in this context may contribute to terminal cell differentiation. The mda-7 gene may also function as a negative regulator of melanoma progression.

The early gene expression changes mediating carcinogen enhancement of viral transformation (CET) remain to be elucidated. A model cell culture system has been developed that is now permitting a molecular analysis of CET. Pretreatment of cloned rat embryo fibroblast (CREF) cells with methyl methanesulfonate (MMS) prior to infection with the cold-sensitive host-range type 5 adenovirus mutant, H5hr1, results in a dose-dependent increase in viral transformation. The present study investigates the role of immediate-early response genes, specifically c-fos, in the CET process. MMS pretreatment, alone or in combination with infection with H5hr1 temporally and differentially increases c-fos, c-jun, jun-B, jun-D and c-myc steady-state mRNA levels. Maximum induction occurs with c-fos and c-jun 8 to 12 h posttreatment and the magnitude of response is generally greatest in CREF cells pretreated with MMS and then infected with H5hr1. Enhancement in RNA levels is observed in the presence of cycloheximide indicating that ongoing protein synthesis is not required for induction of c-fos, c-jun, jun-B or c-myc expression. Nuclear run-on analysis indicates an enhancement in transcriptional rates for c-fos, c-jun, jun-B and c-myc in CREF cells treated with MMS or MMS plus infection with H5hr1. A requirement for elevated c-fos in the early stages of CET is indicated by the ability of c-fos antisense oligonucleotides to prevent the CET process. Direct evidence implicating early increases in c-fos as a mediator of the CET process is demonstrated by stably expressing mouse mammary tumor virus promoter-regulated human sense and antisense c-fos genes in CREF cells. Induction of c-fos sense expression by dexamethasone (DEX) in the absence of MMS treatment results in enhanced c-fos mRNA, Fos protein, AP-1 DNA-binding activity and H5hr1-induced transformation and CET. Induction of c-fos expression by DEX in stable c-fos-sense CREF constructs also results in elevated levels of c-jun, jun-B and c-myc mRNA and protein. Conversely, induction of c-fos antisense expression prevents the increase in c-fos mRNA, Fos protein and AP-1 DNA-binding activity and eliminates CET. In the antisense-c-fos constructs, increases in c-jun, jun-B and c-myc mRNA and protein normally induced by MMS also are not apparent. Thus, induction or inhibition in c-fos expression affects the level of expression of additional immediate-early response genes, including c-jun, jun-B and c-myc.(ABSTRACT TRUNCATED AT 400 WORDS)

The combination of recombinant human fibroblast interferon (IFN-beta) and the antileukemic compound mezerein (MEZ) induces terminal differentiation with an irreversible loss of proliferative capacity in human melanoma cells. Using subtraction hybridization, cDNAs were identified that display enhanced expression in terminally differentiated and growth arrested human melanoma cells (Jiang and Fisher, 1993; Jiang et al., 1994a). A specific melanoma differentiation-associated (mda) cDNA, mda-6, is described whose expression inversely correlates with melanoma progression and growth. mda-6 is identical to WAF1/CIP1/SDI1 that encodes the M(r) 21,000 protein (p21) that is an inhibitor of cyclin-dependent kinases. Actively growing normal melanocyte, SV40-immortalized human melanocyte and dysplastic nevus cell lines synthesize elevated levels of mda-6 mRNA; whereas, actively proliferating radial and early vertical growth phase primary melanomas as well as metastatic human melanoma cells produce reduced levels of mda-6 mRNA. Treatment of primary and metastatic human melanoma cells with IFN-beta + MEZ results in growth inhibition and an increase in mda-6 expression. mda-6 expression also increases when human melanoma cells are grown to high saturation densities or when grown in serum-free medium. Using anti-p53 and anti-p21 antibodies, an inverse correlation is found between p53 and p21 protein levels during growth arrest and differentiation. Induction of growth arrest and terminal differentiation in H0-1 human melanoma cells by IFN-beta + MEZ results in a temporal decrease in wild-type p53 protein levels with a corresponding increase in p21 levels. In the Matrigel-assisted melanoma progression model, mda-6 expression decreases in early vertical growth phase primary human melanoma cells selected for autonomous or enhanced tumor formation in nude mice. In metastatic human melanoma cells displaying a loss of metastatic potential resulting from introduction of a normal human chromosome 6, mda-6 mRNA levels increase. Taken together, these studies indicate that mda-6 (p21) may function as a negative regulator of melanoma growth, progression and metastasis.

The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expression in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.

Protein kinase C (PKC) is a key component in signal transduction in eucaryotic cells and when specific PKC isoforms are over-expressed in immortal mammalian cells they can induce transformation-associated properties. In the present study we demonstrate that a cloned PKC beta 1 gene can induce an enhanced expression of the transformed phenotype in type 5 adenovirus (Ad5)-transformed rat embryo (RE) cells (clone E11), a process termed transformation progression. E11 cells expressing the PKC beta 1 gene, clone B1/PKC, produce PKC beta 1 mRNA and display enhanced PKC enzymatic activity and binding of [3H]-phorbol-12,13-dibutyrate (PDBu) to cell surface phorbol ester receptors. B1/PKC cells grow with increased efficiency in agar in comparison with parental E11 cells and anchorage-independence is further enhanced in both cell types by addition of the tumor promoting agent 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A single-exposure of B1/PKC cells to 5-azacytidine (AZA), followed by growth in the absence of this demethylating agent, results in B1/PKC-AZA clones which display a stable reversion of the progression phenotype to that of the unprogressed parental E11 clone. Loss of the progression phenotype corresponds with a reduction in PKC beta 1-induced biochemical and cellular changes. In contrast, progression-suppression does not involve an alteration in expression of the Ad5 transforming genes, E1A and E1B, or the endogenous PKC epsilon gene. TPA cannot induce the progression phenotype in B1/PKC-AZA cells, but it can reversibly induce an increase in the transcriptional rate and steady-state mRNA levels of PKC beta 1 and c-jun and it increases AP-1 DNA-binding. These results indicate that the PKC beta 1 gene can serve as a transformation progression-inducing gene in rat embryo cells previously transformed by Ad5 and progression may be mediated by the inactivation by methylation of an AZA-sensitive 'progression suppressor gene(s)'. The suppression process in B1/PKC cells is independent of expression of the Ad5-transforming genes but correlates directly with the reduced expression of the transfected PKC beta 1 gene in AZA-treated B1/PKC cells.

Carcinogenesis requires a complex series of genetic changes often involving multiple oncogenes and the inactivation of multiple tumor-suppressor genes. We presently examined the effect of the Krev-1 tumor-suppressor gene on the tumorigenic and metastatic potential of Ha-ras-transformed cloned rat embryo fibroblast (CREF) cells. Ha-ras-transformed CREF cells are morphologically transformed and anchorage independent; produce reduced levels of nm23-H1 (a putative metastasis-suppressor gene product) and TIMP-1 (tissue inhibitor of metalloproteinase 1) transcripts and mRNA compared with CREF cells; produce increased levels of cripto, 94-kDa gelatinase/type IV collagenase (94-kDa GEL), osteopontin (OPN) and transin/stromelysin transcripts and mRNA compared with CREF cells; and are tumorigenic and metastatic in both nude mice and syngeneic rats. Ha-ras-transformed CREF cells coexpressing the Krev-1 gene display a reversion in cellular phenotype and gene expression to that of untransformed CREF cells. However, Ha-ras/Krev-1-coexpressing CREF cells retain, albeit with extended latency periods, both tumorigenic and metastatic potential that is not related directly to the final level of Ha-ras or Krev-1 mRNA or the Ha-ras p21 transforming protein. Development of metastatic potential is, however, directly correlated with a reduction in nm23-H1 and TIMP-1 transcription and mRNA levels and an enhanced expression of cripto, 94-kDa GEL, osteopontin and transin. In contrast, expression of additional tumor-suppressor genes, such as the RB gene and p53, or genes associated with tumorigenesis in other model systems, such as major excreted glycoprotein (MEP), 72-kDa gelatinase/type IV collagenase (72-kDa GEL), fibronectin (FIB), tenascin and intracellular adhesion molecule 1 (ICAM-1) is not altered in a consistent manner during in vitro transformation suppression or escape from tumorigenic and metastatic suppression. These results indicate that Krev-1 suppression of the Ha-ras-transformed/oncogenic phenotype is associated with a distinct program of gene expression changes manifested by altered rates of transcription and steady-state mRNA levels of specific oncogenic-suppressing and oncogenic-inducing genes. These data support a model of Ha-ras-induced metastasis in CREF cells that involves a direct modulation in the expression/suppression of specific combinations of oncogenic-suppressor genes and metastasis-promoting genes that are regulated coordinately in the process of tumor progression.

Transformation of secondary Sprague-Dawley rat embryo (RE) cells with type 5 adenovirus (Ad5) results in morphologically transformed cells which can undergo a series of sequential changes resulting in enhanced expression of the transformed phenotype, a process termed progression. Selection for a progressed phenotype often occurs after growth in agar or tumor formation in nude mice, and this process is reversible following treatment of cells with 5-azacytidine. In the present study we have analyzed a series of clonal populations of Ad5-transformed RE cells representing different stages in a defined progression lineage. Progression was not associated with alterations in the steady-state levels of mRNA produced by the viral transforming genes, E1A and E1B, or the cellular gene, c-myc. In addition, the tumor-promoting agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), which induces expression of a progressed phenotype in Ad5-transformed RE cells, did not significantly alter the RNA transcription rates of the Ad5 E1A or E1B genes, the TPA-inducible gene TPA-S1 or the TPA-responsive genes Pro1 or protein kinase C. TPA did, however, increase by 1 h the steady-state level of c-fos mRNA, but this effect was similar in both progressed and unprogressed cells. Progression also did not involve a change in the RNA transcription rate of a number of cellular and viral genes, including actin, c-Ha-ras, c-myc, v-fos, erbB, TGF-alpha, TGF-beta, Pro-2, transin, TPA-R1, v-myb and c-mos, or other adenovirus genes in addition to E1A and E1B, including E2A and E4. Immunoblotting analysis using E1B polyclonal antiserum further indicated that progression was not associated with changes in the levels of an Mr 21,000 polypeptide encoded by E1B. Similarly, immunoprecipitation analysis with an Ad2 E1A monoclonal antibody indicated similar levels of the Mr 55,000 and 48,000 E1A polypeptides, as well as coprecipitated proteins of Mr 300,000, 107,000 and 105,000 [which is the retinoblastoma (Rb) protein], in E11 and E11-NMT cells. Immunoprecipitation of cell lysates with a monoclonal antibody specific for the Mr 105,000 Rb protein further demonstrated that progression also was not associated with a change in the level or state of phosphorylation of the Rb protein. However, transfection of a human Rb gene (also containing a neomycin resistance gene) into Ad5-transformed RE cells was more inhibitory, with respect to formation of G418-resistant colonies, in unprogressed than in progressed Ad5-transformed RE cells.(ABSTRACT TRUNCATED AT 400 WORDS)