E1A Mutations Research Articles

541. Oncolytic Effects of Conditionally Replicating Adenoviruses (CRAds) with Mutations in E1A and E1B Regions

Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated replicating adenoviruses (Ads) with different combinations of E1A & E1B mutant genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent Ads. We show here that cytolytic potency of each gene attenuated replicating adenoviruses differed significantly depending on the presence or deletion of E1B55 kDa and E1B19 kDa function. More specifically, among the constructed (Ad-DE1B19, Ad-DE1B55, Ad-DE1B19/55, and Ad-wt), E1B19 kDa-inactivated adenovirus (Ad-DE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. As an additional effort to increase cancer cell-selectivity of replicating adenovirus, we have generated eleven E1A-mutant Ads (Ad-mt|[num]|1 |[sim]||[num]|11) with deletion or substitution in retinoblastoma (Rb) binding sites of E1A. Of these viruses, Ad-mt|[num]|7 demonstrated significantly improved CPE and viral replication in a cancer cell-specific manner. To further increase the cancer cell-specific cell killing effect of Ad-mt|[num]|7 adenovirus, both E1B19 kDa and E1B55 kDa gene was deleted, resulting in an Ad-|[Delta]|B7 Ad. As assessed using CPE assay, MTT assay, and Ad fiber immunoblot analysis, Ad-|[Delta]|B7 exerted markedly enhanced cancer cell-specific killing effect as well as viral replication in comparison to either Ad-mt|[num]|7 or Ad-|[Delta]|E1B19/55. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-DB7. In summary, we have developed replication competent adenoviruses with significantly improved therapeutic profiles for cancer treatment.

1011. Tumor Specific Oncolytic Adenovirus Vector VRX-011 Which Overproduces ADP and Has the E4 Promoter Replaced by the hTERT Promoter

We have previously evaluated the conditionally replicating adenovirus (Ad) vector named KD3 for possible use in anti-cancer therapy. KD3 markedly overexpresses ADP, an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. KD3 also has two E1A mutations that reduce its replication in primary cells but allow efficient replication in cancer cells. We have also reported on a replication-competent vector named VRX-007 which overexpresses ADP and has wild type E1A. VRX-007 showed remarkable early cytopathic effects, early cell lysis, large plaques, increased cell to cell dissemination in cultured cells, enhanced cytolysis of cultured cancer cells, and efficient suppression of tumor growth in animal models. In this report we describe the vector VRX-011, which is similar to VRX-007 except that the Ad E4 promoter has been replaced by the promoter for human telomerase (hTERT). Telomerase is an enzyme involved in the maintenance of chromosomal termini known as telomeres. About 90% of cancer cells have elevated levels of telomerase activity, whereas telomerase activity is not detected in normal somatic cells. Since E4 proteins are required for Ad replication, VRX-011 is expected to replicate in most cancer cell types but not (well) in non-cancerous cells. VRX-011 replicated as well as Ad5 in the majority of cancer cell lines tested and was growth restricted in primary cells. Ad late protein expression was at normal levels in A549 human lung cancer cells but was markedly reduced in primary cells. Intratumor administration of VRX-011 effectively suppressed Hep3B liver carcinoma xenograft growth in nude mice. Systemic administration of VRX-011 by tail vein injection also efficiently suppressed Hep3B xenograft growth in nude mice. Also, intravenous administration of VRX-011 significantly reduced the number and size of A549 cancer cell metastases in nude mice lungs. Single tail vein administration of different PFU doses of VRX-011 in C57B/6 mice showed no toxicity as indicated by liver enzyme levels and animal weights. These in vitro and in vivo data show that VRX-011 has enhanced specific oncolytic efficacy and restriction in primary cells. VRX-011 may be useful in treating different types of human cancer.

818. Adenovirus Replication-Competent Cancer Vectors That Overexpress Adenovirus Death Protein and Are Targeted to Tumors by Mutations in E1A and/or E4 Promoter Substitutions

818. Adenovirus Replication-Competent Cancer Vectors That Overexpress Adenovirus Death Protein and Are Targeted to Tumors by Mutations in E1A and/or E4 Promoter Substitutions

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein.

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G(0) to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1(-) E3(-) replication-defective vector expressing beta-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10(-4) PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.

E1A represses apolipoprotein AI enhancer activity in liver cells through a pRb- and CBP-independent pathway.

The apolipoprotein AI (apoAI) promoter/enhancer contains multiple cis -acting elements on which a variety of hepatocyte-enriched and ubiquitous transcription factors function synergistically to regulate liver-specific transcription. Adenovirus E1A proteins repress tissue-specific gene expression and disrupt the differentiated state in a variety of cell types. In this study expression of E1A 12Sor 13S in hepatoblastoma HepG2 cells repressed apoAI enhancer activity 8-fold. Deletion mapping analysis showed that inhibition by E1A was mediated by the apoAI promoter site B. E1A selectively inhibited the ability of HNF3beta and HNF3alpha to transactivate reporter genes controlled by the apoAI site B and the HNF3 binding site from the transthyretin promoter. The E1A-mediated repression of HNF3 activity was not reversed by overexpression of HNF3beta nor did E1A alter nuclear HNF3beta protein levels or inhibit HNF3 binding to DNA in mobility shift assays. Overexpression of two cofactors known to interact with E1A, pRb and CBP failed to overcome inhibition of HNF3 activity. Similarly, mutations in E1A that disrupt its interaction with pRb or CBP did not compromise its ability to repress HNF3beta transcriptional activity. These data suggest that E1A inhibits HNF3 activity by inactivating a limiting cofactor(s) distinct from pRb or CBP.

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.

Induction of DNA synthesis and apoptosis in cardiac myocytes by E1A oncoprotein.

Beginning during the second half of gestation, increasing numbers of cardiac myocytes withdraw from the cell cycle such that DNA synthesis is no longer detectable in these cells by neonatal day 17 in vivo. The mechanisms that exclude these and other terminally differentiated cells from the cell division cycle are poorly understood. To begin to explore the molecular basis of the barrier to G1/S progression in cardiac myocytes, we used adenoviruses to express wild-type and mutant E1A proteins in primary cultures from embryonic day 20 rats. While most of these cardiac myocytes are ordinarily refractory to DNA synthesis, even in the presence of serum growth factors, expression of wild-type E1A stimulates DNA synthesis in up to 94% or almost all successfully transduced cells. Rather than complete the cell cycle, however, these cells undergo apoptosis. Apoptosis is limited to those cells that engage in DNA synthesis, and the kinetics of the two processes suggest that DNA synthesis precedes apoptosis. Mutations in E1A that disable it from binding Rb and related pocket proteins have little effect on its ability to stimulate DNA synthesis in cardiac myocytes. In contrast, mutants that are defective in binding the cellular protein p300 stimulate DNA synthesis 2.4-4.1-fold less efficiently, even in the context of retained E1A pocket protein binding. In the absence of ElA pocket protein binding, the usual situation in the cell, loss of p300 binding severely decreases the ability of ElA to stimulate DNA synthesis. These results suggest that the barrier to G1/S progression in cardiac myocytes is mediated. at least in part, by the same molecules that gate the G1/S transition in actively cycling cells, and that p300 or related family members play an important role in this process.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

The effect of E1A transfection on MMP‐9 expression and metastatic potential

The expression of MMP-9 in rat embryo fibroblasts (REF) transformed with Ha-ras or with Ha-ras and v-myc is associated with metastatic behavior. In contrast, REF transformed with Ha-ras and the adenovirus E1A genes (E1A) are tumorigenic, do not release MMP-9 and are rarely metastatic. In this report, we establish that E1A expression results in decreased levels of MMP-9 mRNA in an Ha-ras and v-myc transformed cell line and examine which of the functional domains of E1A participate in the inhibition of MMP-9 expression and which contribute to the suppression of metastasis. The metastatic 2.10 REF line, derived by co-transfection with v-myc and Ha-ras, constitutively expresses high levels of MMP-9 (92 kDa gelatinase). Transfection of E1A wild-type plasmids into this cell line eliminates detectable MMP-9 mRNA expression and greatly reduces MMP-9 activity. Transfection of 2.10 with E1A plasmids encoding mutations in conserved region 2 (CR2) retained inhibition of MMP-9 similar to the inhibition seen with wild-type E1A. Transfection with E1A containing mutations in CRI or the amino terminal region diminished, but did not fully inhibit, MMP-9 expression. In contrast, inhibition of MMP-9 was lost in with E1A mutations in CR3. Cells transfected with E1A mutants in CR1, the amino terminal region or CR3 retained metastatic behavior. Our findings delineate the regions of E1A responsible for MMP-9 inhibition and further define the domains of E1A responsible for inhibition of metastasis.

Adenovirus E1B 19-kilodalton protein overcomes the cytotoxicity of E1A proteins

Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.

Genetic dissection of the transactivating domain of the E1a 289R protein of adenovirus type 2

A series of linker-scanning, deletion, and frameshift mutations were made in the pm975 variant of the adenovirus type 2 E1a gene, which expresses only the larger of the two major E1a proteins. Most of these were within the 46-amino-acid segment unique to the larger E1a protein product (the 289R protein), which confers on it the ability to activate in trans the expression of other genes. The mutations were recombined into virus and assayed by in vitro transcription in nuclei isolated from infected cells for their ability to activate the transcription of other viral early genes and of the endogenous hsp70 gene. Mutant E1a proteins from which the 289R-unique segment was removed by deletion or truncation did not completely lose the ability to transactivate by comparison with a virus which makes no E1a at all, indicating that sequences outside this domain are active in the positive regulation of transcription. The E1a mutations tested fell into several classes: those that increased transactivation of virtually all genes, those that severely depressed transactivation of all genes, and those that depressed transactivation only moderately. Each mutation had similar effects on the expression of all transcription units tested, indicating a common process in their transactivation. However, some mutants in the third category decreased transactivation of some induced genes more severely than of others. Such gene-specific defects suggest the existence of subclasses of E1a-responsive transcription units, consistent with the involvement of diverse proteins in the transactivation of different genes. Two specific structural components of the transactivating domain, a putative metal-binding element and a region with high potential for beta-sheet formation at its carboxy-terminus, appear to be important to the transactivation function.

Relationship between the transforming and transcriptional regulatory functions of adenovirus 2 E1a oncogene

The E1a region of adenoviruses encodes two early proteins of 289 amino acids (289R) and 243R from two differentially spliced mRNAs of 13 and 12 S, respectively. These E1a proteins of adenoviruses are multifunctional and have been shown to play an essential role in cellular immortalization and transformation. The E1a gene is also known to regulate the expression of certain viral and cellular genes in a positive or negative manner. To identify the domains of the E1a proteins required for their transformation and transcriptional regulatory functions, we have constructed and analyzed several E1a mutations. A region located between amino acid residues 125 and 127 appears to be essential for cell transformation in cooperation with both E1b and the activated cellular oncogene, T24 Har- ras. Mutation at this region does not affect the ability of E1a to trans-activate the Ad2 early E2 promoter significantly. Our experiments have not revealed the domain of E1a most essential for trans-repression. However, the region between amino acid residues 125 and 127 appears to have a small effect on E1a-mediated repression of the immunoglobulin heavy-chain and polyoma enhancers but has no significant effect on the SV40 enhancer. From our results, it appears that the transformation function of E1a can be dissociated from its transcriptional regulatory functions.

Cis-acting induction of adenovirus transcription

At early times after adenovirus infection, transcription from all early viral promoters requires the expression of a 289 amino acid phosphoprotein encoded in the pre-early transcription unit E1A. Mutations in E1A result in a marked delay in the induction of early viral transcription. To distinguish between possible models that could account for this delayed transcription, superinfection experiments were performed. Cells were first infected with one E1A mutant, incubated until viral transcription was detectable, and then superinfected with a second EIA mutant with distinguishable transcripts from early region 3 (E3). We found that the second virus to infect the cell recapitulated the delayed transcription induction of E3. Therefore, the delayed expression of early regions after infection with E1A mutants is the result of a time-dependent cis-acting modification of the template. Models which can account for these observations and for the function of E1A protein are discussed.