pRb Binding Research Articles

Destabilization of the RB Tumor Suppressor Protein and Stabilization of p53 Contribute to HPV Type 16 E7-Induced Apoptosis

Cells that express the human papillomavirus (HPV) type 16 E7 oncoprotein are predisposed to undergo apoptosis. Transgenic mice that have E7 expression targeted to either the retinal photoreceptor cells or the lens cells exhibit signs of apoptosis in cells attempting to undergo differentiation. We established a cell culture system to study this process and have determined the domains of E7 that are required for predisposing cells to undergo apoptosis in response to growth arrest signals. Regions within the core pRB binding site of E7 were necessary but not sufficient for inducing apoptosis. Residues within the adenovirus conserved region 1 homology domain and the consensus casein kinase II phosphorylation site are also important for this effect on cell viability. Our data also demonstrate that the ability of E7 to induce destabilization of pRB and stabilization of p53 coincides with E7-mediated transformation and apoptosis.

Both conserved region 1 (CR1) and CR2 of the human papillomavirus type 16 E7 oncogene are required for induction of epidermal hyperplasia and tumor formation in transgenic mice.

High-risk human papillomavirus type 16 (HPV-16) and HPV-18 are associated with the majority of human cervical carcinomas, and two viral genes, HPV E6 and E7, are commonly found to be expressed in these cancers. The presence of HPV-16 E7 is sufficient to induce epidermal hyperplasia and epithelial tumors in transgenic mice. In this study, we have performed experiments in transgenic mice to determine which domains of E7 contribute to these in vivo properties. The human keratin 14 promoter was used to direct expression of mutant E7 genes to stratified squamous epithelia in mice. The E7 mutants chosen had either an in-frame deletion in the conserved region 2 (CR2) domain, which is required for binding of the retinoblastoma tumor suppressor protein (pRb) and pRb-like proteins, or an in-frame deletion in the E7 CR1 domain. The CR1 domain contributes to cellular transformation at a level other than pRb binding. Four lines of animals transgenic for an HPV-16 E7 harboring a CR1 deletion and five lines harboring a CR2 deletion were generated and were observed for overt and histological phenotypes. A detailed time course analysis was performed to monitor acute effects of wild-type versus mutant E7 on the epidermis, a site of high-level expression. In the transgenic mice with the wild-type E7 gene, age-dependent expression of HPV-16 E7 correlated with the severity of epidermal hyperplasia. Similar age-dependent patterns of expression of the mutant E7 genes failed to result in any phenotypes. In addition, the transgenic mice with a mutant E7 gene did not develop tumors. These experiments indicate that binding and inactivation of pRb and pRb-like proteins through the CR2 domain of E7 are necessary for induction of epidermal hyperplasia and carcinogenesis in mouse skin and also suggest a role for the CR1 domain in the induction of these phenotypes through as-yet-uncharacterized mechanisms.

Accumulation of p53 induced by the adenovirus E1A protein requires regions involved in the stimulation of DNA synthesis.

It has been known for some time that expression of the 243-residue (243R) human adenovirus type 5 (Ad5) early region 1A (E1A) protein causes an increase in the level of the cellular tumor suppressor p53 and induction of p53-dependent apoptosis. Deletion of a portion of conserved region 1 (CR1) had been shown to prevent apoptosis, suggesting that binding of p300 and/or the pRB retinoblastoma tumor suppressor and related proteins might be implicated. To examine the mechanism of the E1A-induced accumulation of p53, cells were infected with viruses expressing E1A-243R containing various deletions which have well-characterized effects on p300 and pRB binding. It was found that in human HeLa cells and rodent cells, complex formation with p300 but not pRB was required for the rise in p53 levels. However, in other human cell lines, including MRC-5 cells, E1A proteins which were able to form complexes with either p300 or pRB induced a significant increase in p53 levels. Only E1A mutants defective in binding both classes of proteins were unable to stimulate p53 accumulation. This same pattern was also apparent in p53-null mouse cells coinfected by Ad5 mutants and an adenovirus vector expressing either wild-type or mutant human p53 under a cytomegalovirus promoter, indicating that the difference in importance of pRB binding may relate to differences between rodent and human p53 expression. The increase in p53 levels correlated well with the induction of apoptosis and, as shown previously, with the stimulation of cellular DNA synthesis. Thus, it is possible that the accumulation of p53 is induced by the induction of unscheduled DNA synthesis by E1A proteins and that increased levels of p53 then activate cell death pathways.

P300 binding by E1A cosegregates with p53 induction but is dispensable for apoptosis.

E1A expression during adenovirus infection induces apoptosis. E1A expression causes accumulation of the p53 tumor suppressor protein, and E1A-induced apoptosis is p53 mediated in primary rodent cells, implying that p53 induction may be linked to apoptosis induction by E1A. Adenoviruses containing mutations in the E1A gene were tested for the ability to trigger both p53 accumulation and the appearance of enhanced cytopathy (cyt phenotype) and degradation of DNA (deg phenotype), indicative of apoptosis in infected HeLa cells. The adenoviruses had mutations which disrupted the pRb- and/or p300-binding activities of E1A so that the relationship between p53 induction and apoptosis and binding to these cellular proteins by E1A could be determined. An E1A mutation that specifically disrupted the p300-binding activity failed to induce p53 accumulation, whereas mutations in E1A which affected pRb binding induced p53 accumulation. Thus, p300 binding was required and pRb binding was dispensable for E1A-mediated accumulation of p53 in HeLa cells. All the E1A mutant viruses, regardless of the ability to induce p53 accumulation, induced the cyt and deg phenotypes, suggesting that p53 induction in infected HeLa cells was not essential for apoptosis, nor was binding of E1A to the pRb and/or p300 protein. The possibility that E1A induced a p53-independent apoptosis pathway was tested by analyzing the appearance of the cyt and deg phenotypes in Saos-2 cells, which were null for both alleles of p53, upon adenovirus infection. An adenovirus expressing wild-type 12S E1A induced both the cyt and deg phenotypes in Saos-2 cells, as did all the E1A mutant viruses. Thus, E1A expression during infection of human cells may trigger redundant p53-independent and -dependent apoptotic pathways.

Polyomavirus large T antigen overcomes p53 dependent growth arrest.

Polyomavirus transforms cells in culture and induces tumors in mice without apparent interaction with or inactivation of the p53 tumor suppressor protein. In this report we investigate the ability of polyomavirus T antigens to overcome the growth suppression function of p53. A temperature sensitive p53 gene was introduced into mouse embryo fibroblasts derived from a p53 null mouse, resulting in expression of a protein with a mutant conformation at 37 degrees C and a functionally wild-type conformation at 32 degrees C. We found that expression of p53 at 32 degrees C induced the cyclin-dependent kinase inhibitor p21/WAF1 and arrested cell growth in the G1/G0 phase of the cell cycle. Only the under-phosphorylated form of the retinoblastoma tumor suppressor protein (pRB) was detected in these growth arrested cells. We introduced both polyomavirus large T (LT) and middle T (MT) antigens into this cell line and showed that LT overcame p53-dependent growth arrest, while MT did not. In cells grown at 32 degrees C, LT expession led to cell proliferation and phosphorylation of pRB in the presence of p21. A mutant LT containing a defective pRB binding domain failed to overcome the growth arrest, indicating that interaction of LT with RB proteins is required to override p53 function. Although the polyomavirus T antigens do not interact with p53 directly, our results indicate that the virus, through LT, is able to interfere with the growth suppressive activity of p53.

A DNA polymerase α accessory protein exhibits structural and functional similarities to SV40 large tumor antigen

Untransformed cells have been proposed to require a protein homologous to SV40 large tumor antigen (TAg) which functions as a component of the replicase complex during the initiation of DNA synthesis. By definition, this should be a phosphoprotein which interacts with the retinoblastoma protein (pRb) in G 0 or early G 1, and is capable of binding to and potentiating the activity of DNA polymerase α (pol α). This protein should also be an ATP-dependent helicase which interacts with the single-stranded DNA (ssDNA) binding protein, RP-A. Because of these requirements, a TAg homologous protein could be expected to contain epitopes with amino acid sequences similar to those of TAg at critical functional sites, such as ATP, pRb and pol α binding sites. TAg and a putative cellular homolog of TAg, DNA pol α accessory protein (αAP), were compared for pRb and pol α interaction, and for immunological identity. The analyses utilized immunoaffinity-purified TAg and pRb from a baculovirus expression system, and DNA pol α/primase and αAP chromatographically isolated from a mouse lymphocytic leukemia cell line. Monoclonal antibodies specific for the pol α or pRb binding sites on TAg interacted with αAP strongly enough to be employed for immunoaffinity purification of αAP. Anti-pRb and anti-TAg reciprocally coimmunoprecipitated pRb bound to TAg and pRb bound to αAP. The functional consequences of pol α interaction with TAg or αAP in the presence or absence of pRb was determined using pol α nucleotide incorporation assays. αAP exhibited the capacity to stimulate pol α activity, a capacity which was diminished in the presence of pRb. Lastly, TAg and αAP independently co-purified with pol α through a multi-step chromatographic protocol. These data indicate that a pol α accessory protein, αAP, exhibits functional and immunological similarities to SV40 TAg, suggest that αAP is involved in regulation of the initiation of DNA synthesis, and support the proposal that αAP may be a normal cell protein homologous to SV40 large T antigen.

Distinct domains of adenovirus E1A interact with specific cellular factors to differentially modulate human immunodeficiency virus transcription.

Transcription of human immunodeficiency virus (HIV) type 1 and other viruses is regulated by the transcription factor NF-kappaB, which interacts with the multifunctional cellular protein p300. p300, originally identified by its ability to bind adenovirus early region 1A (E1A), has also been shown to regulate HIV transcription through its interaction with NF-kappaB. The 13S form of E1A activates HIV gene expression, while the 12S form represses its transcription. In this report, we have investigated whether these divergent effects of E1A are dependent upon common or distinct cellular cofactors, including p300, pRb, and the TATA box-binding protein (TBP). Unlike activation in the absence of E1A, cooperative stimulation of HIV gene expression by 13S E1A and RelA was independent of the ability of E1A to bind p300 but was critically dependent on the E1A CR3 region which associates with TBP. In contrast, inhibition of basal HIV gene expression by the 12S form of E1A was dependent on p300 but not pRb or TBP. Interestingly, mutation of the CR2 region of 12S E1A responsible for pRb binding abolished the repression of HIV transcription stimulated by tumor necrosis factor alpha, suggesting that repression of cytokine-activated transcription involves cofactors different from those used in unstimulated cells. Repression and activation of HIV transcription by different forms of E1A are mediated by distinct sets of cellular cofactors. These findings suggest that E1A has evolved to interact by alternative mechanisms with a transcriptional coactivator and its associated cofactors to differentially modulate cellular and viral gene expression.

Polyomavirus large T-antigen binds the ‘pRb related’ protein p130 through sequences in conserved region 2

The transforming potential of DNA tumor viruses derives mainly from the ability of their encoded oncogene products to interact with cellular proteins. Many of these viral oncoproteins share regions of sequence similarity, designated conserved region 1 and 2, which have been implicated in complex formation with pRb, the product of the retinoblastoma tumor suppressor gene, and related p107 and p130 species. It has now been shown that the E1A protein of adenovirus is able to bind to all three pRb-related proteins through sequences in conserved region 1 and 2. We have shown previously that polyomavirus large T-antigen also interacts with pRb and p107 in vitro. The pRb and p107 binding domains reside between residues 141–158, which include conserved region 2. In the present study, we demonstrate that polyomavirus large T-antigen also interacted with p130 in vitro through the same sequences in conserved region 2.

Interaction of rat Cdc37-related protein with retinoblastoma gene product.

By using in vitro binding assays and the yeast two-hybrid system, we have found that a full-length rat Cdc37-related protein (RCdc37) could associate specifically with the retinoblastoma susceptibility gene product (pRB). A series of GST-RCdc37 deletion mutants was constructed to define the amino acid sequence required for the interaction with pRB. A GST-RCdc37 (-20 approximately 229) possessed an activity to associate with pRB, whereas GST-RCdc37 (-20 approximately 165) lost its activity, indicating that the amino acid sequence between 166 and 229 of RCdc37 was essential for the association with pRB. Interestingly, there exists a highly conserved pRB-binding motif (LXCXE; X = any amino acid) that is essential for pRB binding of SV40 large T antigen, E1A, and E7 proteins. A similar experiment using a pRB deletion mutant revealed that the carboxy-terminal portion of pRB was required for binding to RCdc37.

A novel E1A domain mediates skeletal-muscle-specific enhancer repression independently of pRB and p300 binding.

The adenovirus E1A oncoprotein completely blocks muscle differentiation and specifically inhibits the transactivating function of myogenic basic helix-loop-helix (bHLH) transcription factors. This inhibition is dependent on the conserved region CR1 of E1A, which also constitutes part of the binding sites for the pocket proteins pRB, p107, and p130 and the transcriptional coactivators p300 and CBP. Here we report a detailed mutational analysis of E1A and the identification of a muscle inhibition motif within CR1. This motif encompasses amino acids 38 to 62 and inhibits Myf-5- or MyoD-mediated activation of myogenin and the muscle creatine kinase gene. Overexpression of this E1A region also inhibits the conversion of 10T1/2 fibroblasts to the myogenic lineage. The sequence motif EPDNEE (amino acids 55 to 60) within CR1 appears to be particularly important, because point mutations of this sequence diminish the E1A inhibitory activity. Interactions of E1A with pRB and with p300 do not seem to be necessary for the muscle-specific enhancer repression, because E1A mutants which lack these interactions still inhibit Myf-5- and MyoD-mediated transactivation. Moreover, overexpression of p300 fails to overcome muscle-specific inhibition by wild-type E1A and mutant E1A protein which lacks pRB binding. Since we have no evidence for direct E1A interaction with bHLH proteins, we propose that E1A may target a necessary cofactor of the muscle-specific bHLH transcription complex.

Importance of the Ser-132 phosphorylation site in cell transformation and apoptosis induced by the adenovirus type 5 E1A protein.

The 289-residue (289R) and 243R early region 1A (E1A) proteins of human adenovirus type 5 induce cell transformation in cooperation with either E1B or activated ras. Here we report that Ser-132 in both E1A products is a site of phosphorylation in vivo and is the only site phosphorylated in vitro by purified casein kinase II. Ser-132 is located in conserved region 2 near the primary binding site for the pRB tumor suppressor and, in 289R, just upstream of the conserved region 3 transactivation domain involved in regulation of early viral gene expression. Mutants containing alanine or glycine in place of Ser-132 interacted with pRB-related proteins at somewhat reduced efficiency; however, all Ser-132 mutants transformed primary rat cells in cooperation with E1B as well as or better than the wild type when both major E1A proteins were expressed. Such was not the case with mutants expressing only 289R. In cooperation with E1B, the Asp-132 and Gly-132 mutants yielded reduced numbers of smaller transformed foci. With activated ras, all Ser-132 mutants were significantly defective for transformation and the rare foci produced were small and contained extensive areas populated by low densities of flat cells. In the absence of E1B, all Ser-132 mutants induced p53-independent cell death more readily than virus expressing wild-type 289R. These results suggested that phosphorylation at Ser-132 may enhance the binding of pRB and related proteins and also reduce the toxicity of E1A 289R, thus increasing transforming activity.

Functional implications of mutations within polyomavirus large T antigen Rb-binding domain: effects on pRb and p107 binding in vitro and immortalization activity in vivo

In this study, we have extensively modified the Rb-binding domain of polyomavirus large T antigen. Mutant polyomavirus large T antigens were tested for their ability to bind pRb and p107 in vitro and assayed for their capacity to immortalize primary rat embryo fibroblasts in vivo. Polyomavirus large T antigen bound pRb and p107 through a common region located between amino acids 141 to 158, containing the consensus Rb-binding sequence D/N-L-X-C-X-E. Substitution of any amino acid within the core Rb-binding sequence abolished pRb and p107 binding in vitro and immortalization activity in vivo. Substitution of amino acids outside the core Rb-binding sequence reduced pRb and p107 binding in vitro and decreased or abolished immortalization of rat embryo fibroblasts in vivo. Although duplication of the Rb-binding domain within the polyomavirus large T antigen results in a molecule that can bind at least twice as much pRb and p107 in vitro, this mutant displayed an essentially wild-type level of immortalization activity. More importantly, we found that the addition of acidic residues within the casein kinase II consensus phosphorylation region flanking the Rb-binding domain, or the deletion of amino acids 256 to 272, increased the immortalizing activity of the mutant polyomavirus large T antigen. These two mutants displayed a greater than wild-type level of pRb binding in vitro, while in contrast, a decreased affinity for p107 binding in vitro was observed. Together, these results indicate that while pRb binding appears to be an essential event for immortalization, there is no tight correlation between the frequency of immortalization and the absolute level of pRb binding in vitro, indicating that other large T antigen functions are important for cellular immortalization.

The human papillomavirus type 16 E7 gene product interacts with and trans-activates the AP1 family of transcription factors.

The E7 gene product of human papillomavirus type 16 (HPV16) binds to the retinoblastoma gene product (pRb) and dissociates pRb-E2F complexes. However, the observation that the ability of E7 to bind pRb is not required for the HPV16-induced immortalization of primary keratinocytes prompted a search for other cellular factors bound by E7. Using a glutathione-S-transferase (GST) fusion protein system, we show that E7 complexes with AP1 transcription factors including c-Jun, JunB, JunD and c-Fos. The ability of E7 to complex with c-Jun in vivo is demonstrated by co-immunoprecipitation and the yeast two-hybrid system. An analysis of E7 point mutants in the GST system indicates that the E7 zinc-finger motif, but not the pRb binding domain, is involved in these interactions. Using c-Jun deletion mutants, E7 binding maps between amino acids 224 and 286 of c-Jun. E7 trans-activates c-Jun-induced transcription from a Jun responsive promoter, and this activity correlates with the ability of E7 mutants to bind Jun proteins. Finally, a transcriptionally inactive c-Jun deletion, which can bind E7, interferes with the E7-induced transformation of rat embryo fibroblasts in cooperation with an activated ras, indicating that the Jun-E7 interaction is physiologically relevant and that Jun factors may be targeted in the E7 transformation pathway.

CDNA Sequence and Chromosomal Localization of a Novel Human Protein, RBQ-1 (RBBP6), That Binds to the Retinoblastoma Gene Product

We have previously isolated cDNA of a novel protein (RBQ-1, HGMW-approved symbol RBBP6) that binds to the retinoblastoma gene product (pRB). Total nucleotide sequence of the cDNA has now been determined. It encoded a protein of 140 kDa that consists of 948 amino acids and contains multiple repeated sequences like SRS, YRE, and VPPP. The region used for pRB binding was identified on a small region near the C-terminus. We have mapped this gene to 16p11.2–p12 using polymerase chain reaction analysis on a human–hamster hybrid cell panel and chromosomal fluorescencein situhybridization.

70-kDa Heat Shock Cognate Protein Interacts Directly with the N-terminal Region of the Retinoblastoma Gene Product pRb: IDENTIFICATION OF A NOVEL REGION OF pRb-MEDIATING PROTEIN INTERACTION

Retinoblastoma protein (pRb) functions as a tumor suppressor, and certain proteins are known to bind to pRb in the C-terminal region. Although the N-terminal region of pRb may also mediate interaction with some proteins, no such protein has been identified yet. We demonstrated previously the in vivo protein association between pRb and 73-kDa heat shock cognate protein (hsc73) in certain human tumor cell lines. In this report we analyzed the interaction between these two proteins in vitro. Our data showed that hsc73 interacts with the novel N-terminal region of pRb; that is, pRb binds directly to hsc73 and dissociates from hsc73 in an ATP-dependent manner. By using deletion mutants of cDNA encoding pRb, the hsc73 binding site of pRb was determined to be located in the region (residues 301-372) outside the so-called A pocket (residues 373-579) of this tumor suppressor protein. This finding was compatible with the fact that the adenovirus E1A oncoprotein, which is known to bind to the E2F binding pocket region of pRb, could not compete with hsc73 for the binding. Furthermore, phosphorylation of pRb by cyclin-dependent kinase inhibited the binding of pRb to hsc73. These data suggest that hsc73 may act exclusively as the molecular chaperone for nonphosphorylated pRb. As a result, hsc73 may function as a molecular stabilizer of nonphosphorylated pRb.

In Vivo Association of E2F and DP Family Proteins

The mammalian transcription factor E2F plays an important role in regulating the expression of genes that are required for passage through the cell cycle. This transcriptional activity is inhibited by association with the retinoblastoma tumor suppressor protein (pRB) or its relatives p107 and p103. The first cDNA from the E2F family to be cloned was designated E2F-1, and multiple E2F family members have now been identified. They bind to DNA as heterodimers, interacting with proteins known as DP. Here we demonstrate that DP is also a family of polypeptides with at least two members (hDP-1 and hDP-2). Both hDP-1 and hDP-2 bind to all E2F family members in vivo, and each complex is capable of activating transcription. However, the various E2F/DP complexes display strong differences in the ability to bind to either pRB or p107 in vivo, and the specificity of pRB or p107 binding is mediated by the E2F subunit.

In Vivo Viability of Postmitotic Purkinje Neurons Requires pRb Family Member Function

The product of the retinoblastoma susceptibility gene, pRb, is known to be an important regulator of cell division. Disrupted central nervous system development in RB null mice suggests a critical function for pRb in the proliferative arrest and initiation of terminal differentiation of certain neurons. Previously, we have shown that SV40 T-ag expression targeted to Purkinje neurons in transgenic mice causes cell-specific death. Here we describe that T-ag expression induces DNA synthesis and results in DNA fragmentation in Purkinje neurons. Characterization of transgenic mouse lines expressing mutant T-ags demonstrate that the pRb binding domain of T-ag is required for induction of Purkinje cell loss. These findings indicate that a pRb function is required well beyond the completion of Purkinje neuron differentiation and provide a link between cell cycle regulation and neurodegeneration in vivo.

Multiple members of the E2F transcription factor family are the products of oncogenes.

The retinoblastoma gene product (pRB) is a known tumor suppressor, capable of arresting growth in mid-to-late G1. Part of its growth suppression action arises from interaction(s) with one or more members of the E2F family of transcription factors. These proteins most likely contribute to progression from G0 to S phase in mammalian cells, and pRB binding most likely inhibits aspects of their suspected growth-promoting function. Given their growth-stimulating potential, we asked whether one or more E2F alleles can function as oncogenes. Uncloned pools of NIH 3T3 cells producing the pRB binding target E2F-1, E2F-2, or E2F-3 grew in semisolid medium. In addition, they grew to much higher saturation density than controls. From the study of cells producing selected E2F-1 mutant species, it appears that E2F DNA-binding function contributes to, and pRB/E2F binding suppresses, soft-agar growth. Thus, three E2F family members can act as oncogene products, suggesting that part of the normal role of pRB is to down-modulate this potential activity.

Positive and negative regulation of cell proliferation by E2F-1: influence of protein level and human papillomavirus oncoproteins.

E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.

Positive and Negative Regulation of Cell Proliferation by E2F-1: Influence of Protein Level and Human Papillomavirus Oncoproteins

E2F-1 is a member of a family of transcription factors implicated in the activation of genes required for the progression through the S phase of the cell cycle. We have examined the biological activities of E2F-1 with short-term colony-forming assays and long-term immortalization assays. High levels of E2F-1, produced by transfection of the E2F-1 cDNA under the control of a strong promoter, reduced colony formation in normal human foreskin keratinocytes (NHFKs). This inhibition could not be overcome by wild-type human papillomavirus type 16 (HPV16) E6 and E7, two proteins which cooperate to immortalize NHFKs, or by a transdominant p53 mutant. High levels of E2F-1 also inhibited growth of primary and established fibroblasts. The growth-inhibitory activity required the DNA binding function of E2F-1 but not its transactivation or pRB binding activities. A positive role for lower levels of E2F-1 in NHFK immortalization was established by examining the ability of E2F-1 to complement HPV16 E7 mutants that were unable to cooperate with HPV16 E6 to immortalize NHFKs. Although E2F-1 was unable by itself to cooperate with E6, it did, in conjunction with E6, complement a p24GLY mutant of E7 that is defective for immortalization and binding of pRB and pRB-related proteins. By contrast, E2F-1 was unable to complement two other E7 mutants, p2PRO and p31/32ARG/PRO, which are also defective in the immortalization assay, although their proteins display wild-type binding of pRB in vitro. Since the binding of E7 to pRB results in disruption of pRB-E2F interaction and release of transcriptionally active E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E2F, the data support the hypothesis that binding of pRB by E7 and the consequence increase in E3F activity are important but not sufficient for E7-induced keratinocyte immortalization.