Expression Of E1B Genes Research Articles

Impact of E1 and Cre on Adenovirus Vector Amplification: Developing MDCK CAV-2-E1 and E1-Cre Transcomplementing Cell Lines

Adenovirus vectors have been extensively studied through the manipulation of viral genome. However, little attention is being paid to their producer cell-lines; cells are selected according to virus yields, neglecting the expression profile of transcomplementing gene products underlying cell performance. This work evaluates the impact of E1 (E1A and E1B) and Cre recombinase levels in the production of E1-deleted and helper-dependent canine adenovirus type 2 (CAV-2) vectors using MDCK cells. E1A and E1B gene expression and Cre activity were evaluated in different cell clones and compared with the corresponding cell productivity and susceptibility to oxidative stress injury. CAV-2 production was proportional to E1A expression (the highest levels of E1A corresponding to productivities of 3000–5000 I.P./cell), while E1B prolonged host cell viability after infection, conferring protection against apoptosis. Cre recombinase counteracted E1B anti-apoptotic properties, however viral production was maintained under high levels of Cre. Yet, Cre recombinase side effects can be reduced using cell lines with lower Cre-activities, without compromising the excision efficiency of helper vector packaging signal. These results highlight the influence of transcomplementing gene products on CAV-2 producer cell line performance, and the ability to express high levels of E1A and E1B as an important feature for cell line establishment and high adenovirus titers.

A novel triple-regulated oncolytic adenovirus carrying PDCD5 gene exerts potent antitumor efficacy on common human leukemic cell lines

PDCD5 (programmed cell death 5) accelerates apoptosis of certain tumor cells and the replication-defective Ad-PDCD5 may be a promising agent for enhancing chemosensitivity. In this study, a triple-regulated conditionally replicating adenoviruses (CRAd) carrying PDCD5 gene expression cassette, SG611-PDCD5, was engineered. In SG611-PDCD5, the E1a gene with a deletion of 24 nucleotides within CR2 region is controlled under the human telomerase reverse transcriptase (hTERT) promoter, the E1b gene expression is directed by the hypoxia response element (HRE), whereas the PDCD5 gene is controlled by the cytomegalovirus promoter. The tumor-selective replication of this virus and its antitumor efficacy were characterized in several leukemic cell lines in vitro and in xenograft models of human leukemic cell line in nude mice. It was found by RQ-RT-PCR assay that SG611-PDCD5 expressed PDCD5 efficiently in leukemic cells. In K562 tumor xenograft models, SG611-PDCD5 displayed a tumor killing capacity. At a dose of 1 x 10(9) plaque-forming units, SG611-PDCD5 alone could completely inhibit the tumor growth and more effective than replication-defective Ad-PDCD5. Histopathologic examination revealed that SG611-PDCD5 administration resulted in leukemic cell apoptosis. We concluded that the triple-regulated SG611-PDCD5, as a more potent and safer antitumor therapeutic, could provide a new strategy for leukemia biotherapy.

A novel triple-regulated oncolytic adenovirus carrying p53 gene exerts potent antitumor efficacy on common human solid cancers

Conditionally replicating adenoviruses (CRAd) can replicate specifically in cancer cells and lyse them. The CRAds were widely used in the preclinical and clinical studies of cancer therapy. We hypothesize that more precisely regulated replication of CRAds may further improve the vector safety profile and enhance its antitumor efficacy. Here, a triple-regulated CRAd carrying p53 gene expression cassette, SG600-p53, was engineered. In SG600-p53, the E1a gene with a deletion of 24 nucleotides within CR2 region is controlled under the human telomerase reverse transcriptase (hTERT) promoter, the E1b gene expression is directed by the hypoxia response element (HRE), whereas the p53 gene is controlled by the cytomegalovirus promoter. The precise triple-regulation endows SG600-p53 with enhanced antitumor potential and improved safety profile. The tumor-selective replication of this virus and its antitumor efficacy were characterized in several tumor cell lines in vitro and in xenograft models of human non-small cell lung cancer in nude mice. With the selective replication and oncolysis, it was found by ELISA assay that SG600-p53 expressed p53 efficiently in cancer cells. In NCI-H1299 tumor xenograft models, SG600-p53 displayed a tumor-selective killing capacity. At a dose of 2 x 10(9) plaque-forming units, SG600-p53 could completely inhibit the tumor growth and more effective than replication-defective Ad-p53. Histopathologic examination revealed that SG600-p53 administration resulted in cancer cell apoptosis. We concluded that the triple-regulated SG600-p53, as a more potent and safer antitumor therapeutic, could provide a new strategy for cancer biotherapy.

Transcriptional targeting modalities in breast cancer gene therapy using adenovirus vectors controlled by α-lactalbumin promoter

The breast-specific antigen alpha-lactalbumin is expressed in >60% of breast cancer tissues. To evaluate the effect of gene therapy for breast cancer by controlling adenovirus replication with human alpha-lactalbumin promoter, we investigated the activity of a 762-bp human alpha-lactalbumin promoter. Alpha-lactalbumin promoter showed significantly higher activity in MDA-MB-435S and T47D breast cancer cells than in normal breast cell lines or other tumor cell lines. We then developed two novel breast cancer-restricted replicative adenoviruses, AdALAE1a and AdE1aALAE1b. In AdALAE1a, expression of adenoviral E1a gene is under the control of alpha-lactalbumin promoter, and in AdE1aALAE1b, expression of both E1a and E1b genes is under the control of a single alpha-lactalbumin promoter. Both breast cancer-restricted replicative adenoviruses showed viral replication efficiency and tumor cell-killing capability similar to wild-type adenovirus in MDA-MB-435S and T47D cells. The replication efficiency and tumor cell-killing capability of both viruses were attenuated significantly in cells that did not support alpha-lactalbumin promoter. AdE1aALAE1b showed better breast cancer-restricted replication than AdALAE1a, suggesting that a transcriptional targeting modality with alpha-lactalbumin promoter controlling both E1a and E1b gene expression is superior to alpha-lactalbumin promoter controlling only E1a gene expression. Importantly, we found that AdE1aALAE1b could be used to target hormone-independent breast tumors in vivo by inhibiting the growth of MDA-MB-435S s.c. tumors. These data showed that alpha-lactalbumin promoter could regulate the replication of adenovirus to target hormone-independent breast cancers, suggesting that alpha-lactalbumin promoter can be used to develop a novel therapeutic modality for hormone-independent breast cancer.

Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell line, A549, derived from lung carcinoma

Human adenovirus type 40 (Ad40) cannot propagate in conventional established human cell lines such as KB or HeLa cells. However, it has been shown that Ad40 DNA replicates in KB18 cells which express Ad2 E1B genes, suggesting that Ad40 is defective in the E1B gene function in KB or HeLa cells. We show here that Ad40 can propagate and produce plaques on A549 cells which do not contain Ad E1B genes. Our experiments show that the levels of replication of Ad40 DNA and production of infectious Ad40 virus in A549 cells are the same as or higher than those in 293 or KB18 cells. Dot blot analysis shows that the levels of Ad40 E1A and E1B mRNAs expressed in A549 cells at early to intermediate times postinfection are at least 10-fold higher than those in KB or KB18 cells. Northern (RNA) blot analysis shows that large E1B mRNA species (approximately 24S to 26S) are synthesized prior to the onset of DNA replication in A549 cells. No E1B mRNA species are synthesized in KB or KB18 cells at early times postinfection, and no differences in the expression of E1B mRNAs are seen between KB and KB18 cells. The experiment suggests that A549 cells have a cellular factor(s) which activates Ad40 E1B mRNA synthesis and that the E1B mRNA synthesis helps Ad40 propagation. In contrast, Ad40 can propagate in KB18 cells by using Ad2 E1B gene products that are constitutively expressed in this cell line. Furthermore, this result shows that Ad40 cannot propagate in KB cells because of the failure in the expression of E1B genes at early times postinfection.

The efficiency of adenovirus transformation of rodent cells is inversely related to the rate of viral E1A gene expression

While the products of the type 5 adenovirus E1A and E1B genes can initiate pathways leading to a transformed rodent cell, little is known about how the rate of viral early gene expression influences the efficiency of this process. An adenovirus mutant [E1a(r) virus] that expresses its viral E1A and E1B genes at as much as a 100-fold-reduced rate relative to wild-type virus in infected CREF or HeLa cells transforms CREF cells at an 8-fold-higher efficiency than wild-type virus. Additional studies show that the reduction in viral E1A gene expression is solely responsible for this transformation phenotype, and at this low rate of viral E1A gene expression both E1A gene products must be expressed. Unlike previously characterized viruses which transform CREF cells at frequencies greater than wild-type virus, the foci obtained following E1a(r) virus infection were indistinguishable from those arising from wild-type virus by several criteria (morphological characteristics and anchorage-independent growth). Surprisingly, an analysis of viral early gene expression from a panel of wild-type- and E1a(r) virus-transformed CREF cell lines showed similar average rates of both viral E1A and E1B gene expression. By using an adenovirus-transformed cell line that is cold-sensitive for maintenance of the transformed cell phenotype, we show that both wild-type and the E1a(r) viruses can transform these cells at equally high efficiencies at the nonpermissive temperature of 32 degrees C. Our findings suggest that the process leading to a fully transformed cell involves multiple stages, with an early stage being facilitated by a reduced rate of viral E1A gene expression.

Expression of the E4 gene is required for establishment of soft-agar colony-forming rat cell lines transformed by the adenovirus 12 E1 gene.

Rat 3Y1 cells were transfected with recombinant gARC ( pSV2gpt carrying the adenovirus 12 early region 1 [E1] gene), and focus formation was observed in monolayer cultures after culture of cells in gpt-selective medium (Eagle medium containing 10% fetal calf serum, xanthine, thymidine, aminopterin, and mycophenolic acid) for 10 days, followed by focus formation. Transformed E1Y cell lines were then established from these foci. The E1Y cells were transformed morphologically similarly to cells transformed with intact adenovirus 12 DNA but formed no colonies in soft-agar culture and induced tumors in transplanted rats only after a long incubation period. For the establishment of completely transformed cells, 3Y1 cells were transformed with combinations of gARC , pE3 (pBR322 carrying the adenovirus 12 E3 gene), and gE4 ( pSV2gpt carrying the adenovirus 12 E4 gene) DNA. E1- 3Y cells (3Y1 cells transformed with gARC and pE3 DNA), E1- 4Y cells (3Y1 cells transformed with gARC and gE4 DNA), and E1-3- 4Y cells (3Y1 cells transformed with gARC , pE3 , and gE4 DNA) were established. These transformed cell lines were compared for growth in Eagle medium with 2 or 10% fetal calf serum, colony formation in soft-agar culture, and tumor growth in rats transplanted with the transformed cells. Several transformed cell lines of E1- 4Y and E1-3- 4Y cells showed colony formation in soft-agar culture and abundant expression of the E1B gene. T antigen f was seen by immunofluorescence as flecks in these cells, in which the E4 gene was transcribed, but was not seen in E1Y cells, suggesting that T antigen f was encoded by the E4 gene. The suggestion was confirmed by the observation that T antigen f was detected in COS-1 cells transfected singly with gE4 DNA by immunofluorescence with polyclonal and monoclonal antibodies. Transcription of the E4 gene was confirmed in gE4 -transfected COS-1 cells. T antigen f, one of the E4 gene products, was identified as a polypeptide of molecular weight 11,000 (E4- 11K ) by immunoprecipitation with monoclonal antibodies. The above results also suggest that expression of the E4 gene gives cells the advantage of forming colonies in soft-agar culture. A tendency was noticed for E1B gene expression to be enhanced by E4 gene expression. The relationship between enhancement of colony formation in soft-agar culture and enhancement of E1B gene expression is discussed.

Deletion and insertion mutations in early region 1a of type 5 adenovirus that produce cold-sensitive or defective phenotypes for transformation.

On the basis of earlier findings showing that H5hr1 (hr1) is cold sensitive for transformation, a series of mutants were constructed so that they contained deletions or insertions in different sites of early region 1a (E1a) to ascertain: (i) whether the cold-sensitive phenotype of hr1 was the result of the identified single-base pair deletion of nucleotide 1,055 or due to a missense mutation at another site and (ii) what region and how much of the E1a 51-kilodalton protein is actually required to produce cell transformation. A mutant, H5dl101 (dl101), was constructed to contain a 5-base pair deletion of nucleotides 1,008 to 1,012, which produced a frameshift and a subsequent stop codon at nucleotide 1,241. This mutant, which should encode a truncated 33-kilodalton protein in place of the wild-type 51-kilodalton protein, had a cold-sensitive phenotype for transformation essentially identical to hr1. Consonant with this finding, a mutant (H5in106) engineered to contain a 16-base pair insertion initiated after nucleotide 1,009, with a stop codon beginning at the newly inserted nucleotide 1,013, also had a cold-sensitive phenotype like hr1 and dl101. It is striking, however, that a mutant (H5dl105) with a 69-base pair deletion beginning at nucleotide 1,003, and having a stop codon at nucleotide 1,544, was totally defective for transformation at any temperature. Transfection studies with plasmids containing the E1a or E1a and E1b genes of sub309, hr1, and dl101 further revealed that the cold-sensitive transformation phenotype observed could be exhibited in the absence of viral E1b gene expression.