AAV2/8-humanFOXP3 gene therapy shows robust anti-atherosclerosis efficacy in LDLR-KO mice on high cholesterol diet.

Inflammation is a key etiologic component in atherogenesis. Previously we demonstrated that adeno-associated virus (AAV) 2/8 gene delivery of Netrin1 inhibited atherosclerosis in the low density lipoprotein receptor knockout mice on high-cholesterol diet (LDLR-KO/HCD). One important finding from this study was that FOXP3 was strongly up-regulated in these Netrin1-treated animals, as FOXP3 is an anti-inflammatory gene, being the master transcription factor of regulatory T cells. These results suggested that the FOXP3 gene might potentially be used, itself, as an agent to limit atherosclerosis. To test this hypothesis AAV2/8 (AAV)/hFOXP3 or AAV/Neo (control) gene therapy virus were tail vein injected into the LDLR-KO/HCD animal model. It was found that hFOXP3 gene delivery was associated with significantly lower HCD-induced atherogenesis, as measured by larger aortic lumen cross sectional area, thinner aortic wall thickness, and lower aortic systolic blood velocity compared with Neo gene-HCD-treated controls. Moreover these measurements taken from the hFOXP3/HCD-treated animals very closely matched those measurements taken from the normal diet (ND) control animals. These data strongly suggest that AAV/hFOXP3 delivery gave a robust anti-atherosclerosis therapeutic effect and further suggest that FOXP3 be examined more stringently as a therapeutic gene for clinical use.

HPV-16 E1, E2 and E6 each complement the Ad5 helper gene set, increasing rAAV2 and wt AAV2 production.

Adeno-associated virus type 2 (AAV) is a popular vector for human gene therapy, because of its safety record and ability to express genes long term. Yet large-scale recombinant (r) AAV production remains problematic because of low particle yield. The adenovirus (Ad) and herpes (simplex) virus helper genes for AAV have been widely used and studied, but the helper genes of human papillomavirus (HPV) have not. HPV-16 E1, E2 and E6 help wild-type (wt) AAV productive infection in differentiating keratinocytes, however, HEK293 cells are the standard cell line used for generating rAAV. Here we demonstrate that the three HPV genes were unable to stimulate significant rAAV replication in HEK293 cells when used alone. However, when used in conjunction (complementation) with the standard Ad5 helper gene set, E1, E2 and E6 were each capable of significantly boosting rAAV DNA replication and virus particle yield. Moreover, wt AAV DNA replication and virion yield were also significantly boosted by each HPV gene along with wt Ad5 virus co-infection. Mild-to-moderate changes in rep- and cap-encoded protein levels were evident in the presence of the E1, E2 and E6 genes. Higher wt AAV DNA replication was not matched by similar increases in the levels of rep-encoded protein. Moreover, although rep mRNA was upregulated, cap mRNA was upregulated more. Higher virus yields did correlate most consistently with increased Rep52-, VP3- and VP-related 21/31 kDa species. The observed boost in wt and rAAV production by HPV genes was not unexpected, as the Ad and HPV helper gene sets do not seem to recapitulate each other. These results raise the possibility of generating improved helper gene sets derived from both the Ad and HPV helper gene sets.

Systemic human Netrin-1 gene delivery by adeno-associated virus type 8 alters leukocyte accumulation and atherogenesis in vivo.

Atherosclerosis is an inflammatory disorder of arteries. Atherosclerotic plaque, in its early to intermediate stages, is composed largely of lipid-engorged foam cells. These foam cells are derived from the trafficking of monocytes (Mo) into the arterial intima, attracted to the site by chemoattractants. Given that foam cells are derived from the trafficking of Mo, the use of Netrin-1, an Mo chemorepellent, may be useful in limiting Mo accumulation and subsequent plaque formation. To investigate the potential of Netrin-1 for limiting atherosclerosis, we systemically delivered its human (h) cDNA by adeno-associated virus type 8 (AAV8, single-stranded structure) delivery into low-density lipoprotein receptor knockout (LDLR-/-) mice and placed the animals on a high cholesterol diet (HCD). Compared with control neomycin resistance (Neo) gene delivery/HCD, hNetrin-1 delivery resulted in a significant reduction in plaque formation, as determined by larger aortic lumen size, thinner intima-media thickness and lower blood velocity than the Neo/HCD control (all statistically significant). Indices of monocyte/macrophage (Mo/MΦ) accumulation, CD68, integrin, alpha M (ITGAM) and egf-like module containing, mucin-like, hormone receptor-like 1 (EMR-1), were reduced in hNetrin-1/HCD-treated animal's aortas and spleens compared with Neo/HCD-treated animals. Unexpectedly, CD25 and foxp3 (regulatory T cells (Tregs)) in the aorta were strongly upregulated. This is the first time the Mo/MΦ chemorepellent approach, and specific Netrin-1 gene delivery, has been performed for the reduction of Mo/MΦ burden and atherosclerosis. In addition, Netrin-1 has never before been linked to altered Treg levels. These data strongly suggest that hNetrin-1 gene delivery can reduce Mo/MΦ accumulation, inflammation and subsequent plaque formation.

Overexpression of TGFbeta1 by adeno-associated virus type-2 vector protects myocardium from ischemia-reperfusion injury.

Transforming growth factor beta(1) (TGFbeta(1)) has been purported to protect tissues from ischemia-reperfusion (I-R) injury. This study was designed to examine if overexpression of TGFbeta(1) using adeno-associated virus type 2 (AAV) protects cardiomyocytes from reoxygenation injury. TGFbeta(1) was overexpressed in cultured HL-1 mouse cardiomyocytes by transfection with AAV/TGFbeta(1)(Latent) or with AAV/TGFbeta(1)(ACT) (active TGFbeta(1)). TGFbeta(1) upregulation reduced cardiomyocyte apoptosis and necrosis induced by 24 h of hypoxia followed by 3 h of reoxygenation concomitant with reduction in reactive oxygen species release, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NF-kappaB expression. Transfection with AAV/TGFbeta(1)(ACT) was superior to that with AAV/TGFbeta(1)(Latent). To determine if AAV/TGFbeta(1)(ACT) upregulation in vivo would induce cardioprotection from I-R injury, rat hearts were injected with AAV/TGFbeta(1)(ACT) or phosphate-buffered saline (PBS). Six weeks later, TGFbeta(1)(ACT) was upregulated throughout the myocardium. Following I-R, AAV/TGFbeta(1)(ACT)-overexpressing rats had much smaller infarct size (P<0.01 vs PBS group), which was also related to reduced activation of NADPH oxidase and NF-kappaB, and lower levels of malondialdehyde in I-R tissues. These data demonstrate that overexpression of TGFbeta(1) by AAV can protect cardiac tissues from reperfusion injury, possibly via antioxidant mechanism. These findings suggest potential of TGFbeta(1)(ACT) gene therapy for cardioprotection from I-R injury.

Multiple human papillomavirus types replicate in 3A trophoblasts.

Human papillomavirus (HPV) are more prevalent in spontaneous abortions than elect abortions and preferentially infect the trophoblasts. Related to this, HPV type 16 has been shown to productively replicate in 3A trophoblasts in tissue culture. Extending these earlier studies, the described study addresses the issue whether other genital HPV types (11, 18, and 31) can replicate in trophoblasts. In determining this, HPV-11, 18, or 31 genomic DNAs were lipofected into 3A trophoblasts in culture, thus finding all three HPV types could de novo DNA replicate in 3A trophoblasts (Southern blot) and sequentially express their early and late genes as RNA (RT-PCR) and as protein (immunohistochemistry for L1). HPV-transfected 3A lysates from all three HPV types were also shown to contain HPV infectious units by infection of normal skin raft cultures and by neutralization by specific antibody. Furthermore, microarray analysis revealed the gene expression profile of normal keratinocytes (NK) was closer to 3A trophoblasts than to normal fibroblasts. Moreover, the critical HPV transcription factors AP-1 and Sp1 were found to be more highly expressed in 3A cells than NK. These findings suggest trophoblasts, like squamous epithelium, are broadly permissive for HPV, and some similarities in the gene expression repertoire of these two cell types are consistent with this. Finally, these data support our previous results that demonstrate the relationship between HPV infection of the trophoblast and spontaneous abortions.

Inhibition of H-ras expression by the adeno-associated virus Rep78 transformation suppressor gene product.

Here the adeno-associated virus Rep78 gene product was found to inhibit the expression of the chloramphenicol acetyltransferase and the bladder cancer-derived EJ-H-ras coding sequences when they were under the control of the natural cellular H-ras regulatory sequences. However, Rep78 had little or no effect on the expression of these same coding sequences when they were under the control of the regulatory sequences of the murine osteosarcoma virus long terminal repeat. These data indicate that the inhibition of H-ras by Rep78 depends upon sequences present within the cellular H-ras upstream regulatory region. Furthermore, these and earlier data indicate that Rep78 functions as an "antioncogene" or transformation suppressor gene, inhibiting H-ras as well as several viral oncogenes.

Adeno-associated virus inhibits human papillomavirus type 16: a viral interaction implicated in cervical cancer.

Human papillomavirus (HPV) infection, in particular that by HPV type 16, is positively associated with cervical/genital cancer. In contrast, human adeno-associated virus (AAV) infection is negatively associated with these same cancers. AAV has also been found to inhibit the oncogenic properties of a variety of DNA viruses, including bovine papillomavirus type 1, a relative of HPV-16. Taken together, these findings suggested the possibility that AAV and HPV-16 might interact, with AAV inhibiting HPV-16's oncogenic phenotype. Here this hypothesis is addressed using tissue culture assays measuring HPV-16-directed phenotypes. It is found that the cotransfection of AAV type 2 Rep78-positive plasmids resulted in the inhibition of HPV-16 sequence containing plasmids in oncogenic transformation/focus formation, G418-resistant colony formation, and chloramphenicol acetyltransferase expression assays. These data are consistent with the hypothesis that AAV's negative association with cervical cancer is at least partially due to its ability to inhibit HPV-16 expression.

The adeno-associated virus Rep78 major regulatory/transformation suppressor protein binds cellular Sp1 in vitro and evidence of a biological effect.

Adeno-associated virus (AAV) Rep78 is a multifunctional protein that is required for AAV transcriptional activity, AAV DNA replication, and possibly for site-specific integration of AAV into human chromosome 19. Rep78 is also able to inhibit a variety of heterologous promoters, including those of c-H-ras, human papillomavirus types 16 and 18, and HIV type 1. However, Rep78 is unable to significantly affect murine osteosarcomavirus (MSV). It was noticed that promoters that are inhibited possess binding motifs for the cellular transcription factor Sp1, whereas the MSV long terminal repeat promoter did not. These data stimulated the hypothesis that Rep78 may recognize and interact with cellular Sp1. Here, we demonstrate that Rep78 is able to interact with Sp1 in vitro as analyzed by West(far)-Western, electrophoretic mobility shift assay-supershift, and coimmunoprecipitation analyses. Furthermore, in support of an in vivo biological effect from this interaction, Rep78 is demonstrated to inhibit a synthetic, Sp1-dependent promoter. Further still, the insertion of Sp1 DNA binding motifs into the Rep78-resistant MSV long terminal repeat results in a promoter that has increased sensitivity to inhibition by Rep78. Finally, it is demonstrated that the Sp1-Rep78 interaction requires the amino half of Rep78. The interaction of Rep78 with Sp1, along with possible downstream effects on the transcription initiation process of RNA polymerase II, may partially explain the rather broad-based antitumor abilities of AAV.

Expression of CD56 by human papillomavirus E7-specific CD8+ cytotoxic T lymphocytes correlates with increased intracellular perforin expression and enhanced cytotoxicity against HLA-A2-matched cervical tumor cells.

Human papillomavirus (HPV) infection represents the most important risk factor for developing cervical cancer. In this study, we examine the potential of full-length E7-pulsed autologous dendritic cells (DCs) to induce antigen-specific CTL responses from the peripheral blood of healthy individuals against HLA-A2-matched HPV-16 and HPV-18-positive tumor target cells in vitro. We show that DCs pulsed with E7 oncoprotein can consistently stimulate antigen-specific CTL responses that recognize and lyse HPV-16 or HPV-18-positive naturally infected cervical cancer cell lines. HPV-negative, EBV-transformed lymphoblastoid cell lines (LCLs) sharing the HLA haplotype of the target tumor cells, as well as autologous donor LCLs, were not significantly killed by E7-specific CTLs. Cytotoxicity against HLA-A2-matched HPV-16 and HPV-18 tumor target cells could be significantly inhibited by anti-HLA class I and by anti-HLA-A2 monoclonal antibodies. CD8+ CTLs expressed variable levels of CD56 and showed a strongly polarized Type 1 cytokine profile. Sorting of the CD8+ T cells on the basis of CD56 expression demonstrated that the most highly cytotoxic CTLs were CD56+ and expressed higher levels of perforin and IFN-gamma, compared with the CD8+/CD56- population. Taken together, these data demonstrate that full-length, E7-pulsed DCs can consistently induce E7-specific CD8+ CTL responses in healthy individuals that are able to kill naturally HPV-16 and HPV-18-infected cancer cells, and that CD56 expression defines a subset of CD8+ CTLs with high cytolytic activity against tumor cells.

AAV2 X increases AAV6 rep/cap-driven rAAV production.

We have recently identified a new gene, involved in DNA replication, at the far 3' end of the adeno-associated virus type 2 (AAV2) genome. The AAV type 6 (AAV6) genome has a disrupted X open reading frame (ORF) whose two halves, when combined, have full-length homology and comparable size to AAV2 X. Hypothesizing that AAV6 X is inactive, we assessed if AAV2 X augments recombinant (r)AAV2 DNA replication and virion production, but with rep and cap trans-functions of AAV6. Using AAV2 X expressing HEK293 cell lines we show AAV2 X significantly boosts rAAV DNA replication/virion production, driven by AAV6 rep/cap as it does the AAV2 rep/cap system. Protein BLAST search for homology between AAV2 X and various AAV Rep78 proteins suggests that X might be AAV8 Rep78-derived and have some of its activities. These data suggest that AAV2 X, and the corresponding X genes of other AAV types/clades, warrant further study.

The adeno-associated virus Rep78 major regulatory protein forms multimeric complexes and the domain for this activity is contained within the carboxy-half of the molecule.

The adeno-associated virus (AAV) encoded Rep78 is a multifunctional protein which is able to regulate transcription, is required for AAV DNA replication, and appears necessary for site specific integration of AAV DNA into human chromosome 19. Being analogous to the large T antigen, the replication protein of polyomaviruses which is known to homo-multimerize, it seemed likely that the Rep78 protein would also interact with itself to carry out at least some of its functions. Furthermore, in electrophoretic mobility shift assay studies by many laboratories on Rep78/68 protein interaction with AAV terminal repeat DNA it has been noticed that multiple high bands often result. These data suggest Rep78-Rep78 interaction. In this study it is directly demonstrated that Rep78 is able to form multimeric complexes as measured by West-Western and chemical cross-linking assays. Furthermore, using an amino-truncated Rep78 protein, it is demonstrated that the Rep78 homo-multimerization domain is contained within the carboxy-half of the protein.

The trans-inhibitory Rep78 protein of adeno-associated virus binds to TAR region DNA of the human immunodeficiency virus type 1 long terminal repeat.

The large rep gene products, Rep78 and Rep68, of adeno-associated virus (AAV) are pleiotropic effector proteins which are required for AAV DNA replication and the trans-regulation of AAV gene expression. Apart from these essential functions prerequisite for the life cycle of AAV, these rep products are able to inhibit the replication and gene expression of human immunodeficiency virus type 1 (HIV-1) and a number of DNA viruses. Here, it is demonstrated that Rep78, as a chimeric with the maltose binding protein, directly binds the full-length HIV-1 long terminal repeat (LTR), and to a subset of these sequences containing the trans-activation response (TAR) sequence as DNA. These interactions, an effector protein physically binding a target promoter, suggest a direct mechanism of action for Rep78 inhibition. Furthermore, competitive binding studies between the TAR region and the full-length HIV-LTR, strongly suggested that another site(s) within the LTR was also bound by Rep78. Finally, as Rep78 binding is also believed to be affected by secondary structure within the DNA, it was found that Rep78 preferentially binds with HIV-LTR sequences with promoted secondary structure generated by heat denaturation and rapid cooling.

The packaging capacity of adeno-associated virus (AAV) and the potential for wild-type-plus AAV gene therapy vectors.

Because of its ability to integrate chromosomally and its non-pathogenic nature, adeno-associated virus (AAV) has significant potential as a human gene therapy vector. Here we investigate the maximum amount of DNA which can be inserted into the AAV genome and still allow efficient packaging into an infectious virus particle. Altered wild-type AAV genomes were constructed with inserts, which increased in size by 100 bp, ligated at map unit 96. These large wild-type-plus genomes were able to replicate and produce infectious virus, at levels slightly reduced but comparable to normal sized wild type, until the insert size reached 1 kb. These data indicate that the maximum effective packaging capacity of AAV is approximately 900 bp larger than wild type, or 119%. Furthermore, it is demonstrated that these large AAV genomes are able to latently infect cells by chromosomal integration as does wild-type AAV. These data suggest that therapy vectors carrying a foreign gene of 900 bp or less can be generated from AAV, by ligation into non-essential locations, and result in a recombinant AAV virus with a fully wild-type phenotype. Such wild-type-plus AAV vectors will have both advantages and disadvantages over defective recombinant AAV virus - the most important advantages being the ease in which high titers of infectious virus can be generated and the ability to specifically integrate within chromosome 19. Once the concern subsides over the presence of wild-type AAV in clinical applications, wild-type AAV vectors may find specific application niches for use in human gene therapy.

Role of the terminal repeat GAGC trimer, the major Rep78 binding site, in adeno-associated virus DNA replication.

The adeno-associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36-16) and includes the 'core' GAGC trimer (GAGC3, nt 33-22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.

Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus.

The genetic manipulation of antigen-presenting dendritic cells (DC) offers promise for stimulating the immune response, in particular for anticancer and antiviral protocols. As adeno-associated virus (AAV) has shown promise as a gene delivery vector for transducing a variety of hematopoietic cell types, we have investigated AAV's ability to genetically alter DC. In this analysis, we modified the standard granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) treatment of adherent monocytes to generate DC. In our protocol, adherent monocytes were first infected with an AAV/GM-CSF/Neo vector, and the addition of IL-4 was delayed for 2 days to allow for a brief period of monocyte proliferation. AAV-mediated transduction of the GM-CSF and Neo genes into monocytes/DC precursors was demonstrated by G418 selection, GM-CSF secretion, GM-CSF RNA expression (reverse transcriptase-polymerase chain reaction amplification [RT-PCR]), and cell proliferation. Cells resulting from infection with AAV/GM-CSF/Neo virus, and subsequent IL-4 and tumor necrosis factor-alpha (TNF-alpha) treatment, displayed multiple classic markers consistent with mature DC. Finally, chromosomal integration of the AAV vector was also demonstrated in sorted CD83+ DC. These data strongly suggest that AAV vectors will be useful for the genetic manipulation of DC and suggest that the transduction of the GM-CSF gene was able to fully replace the need for exogenous GM-CSF in the production of mature DC.

Rapid induction of cytotoxic T-cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector.

We have shown that the pulsing of dendritic cells (DCs) with human papillomavirus type 16 (HPV-16) antigen proteins by lipofection stimulates class I-restricted cytotoxic T lymphocyte (CTL) response against primary cervical cancer cells. Also, we have shown that adeno-associated virus (AAV) was able to effectively deliver a cytokine gene into DCs. It has been our hypothesis that the delivery of antigen genes into DCs, resulting in endogenous and continuous antigen protein expression, may result in an improvement in T-cell priming by DCs. Here, DCs are pulsed (infected) with an AAV vector containing the HPV-16 E6 gene. After infection, transduced E6 gene mRNA expression and vector chromosomal integration could be identified in infected DCs. Furthermore, priming rosettes formed at early times when the AAV/E6 vector was used. Most importantly, AAV/E6 vector pulsing of DCs induced, after only 7 days of priming, a strong CTL response against primary cervical cancer cell lines, compared to bacterial E6 protein lipofection. Killing was significantly blocked by the addition of anti-MHC class I antibodies. Fluorescence-activated cell sorter (FACS) analysis of resulting primed cell populations revealed higher levels of CD8+ T cells by AAV-based pulsing, with little evidence of CD56 (NK). FACS analysis of the DC populations revealed that AAV/E6 vector-pulsed DCs had higher levels of CD80 and lower levels of CD86 than protein-pulsed DCs. These data suggest that rAAV may be appropriate for antigen pulsing of DCs for immunotherapy protocols. Finally, our protocol represents an advance in regards to the time needed for generating a CTL response compared to other techniques.

Effects of concurrent cisplatinum administration during radiotherapy vs. radiotherapy alone on the immune function of patients with cancer of the uterine cervix.

PURPOSE: To compare the effects of concurrent administration of cisplatinum (40 mg/m(2)/weekly) with radiation therapy (C-RT) to those induced by radiation therapy alone (RT) on the immune function of patients with locally advanced cervical cancer. METHODS AND MATERIALS: In 8 prospectively randomized patients (i.e., 4 receiving RT vs. 4 receiving C-RT), lymphocyte populations including CD3+, CD4+ and CD8+ T-cell subsets, B cells (CD19+) and natural killer cells (CD56+, CD16+, CD3-) were studied before, during, and after therapy. Expression of the activation marker CD25 on CD3+ T cells, intracellular levels of perforin in CD8+ and CD56+ cells, and interferon-gamma (IFN-gamma) and IL-2 in CD4+ and CD8+ T cells was also measured. Finally, lymphoblast transformation and natural killer (NK) cytotoxic activity were assessed. RESULTS: Both RT and C-RT significantly decreased the mean absolute number of all lymphocyte subsets compared to pretreatment levels (p > 0.001). However, no differences were detected in the characteristics or the magnitude of the lymphopenia induced by the two treatments. Both RT and C-RT increased similarly the percentages of CD25-positive lymphocytes (p > 0.001), and significantly decreased PHA-induced T-cell lymphoblast transformation (p > 0.001) and NK cytotoxic activity against K562 cells (p > 0.001). The percentage of perforin-positive and CD8+ T cells was not altered during either treatment, whereas the percentage of perforin-positive and CD56+ cells was significantly reduced during both treatments, and correlated with reduced cytotoxicity against K562 cells. The percentages of CD8+ IFN-gamma+ and CD4+ IFN-gamma+ T cells as well as that of CD8+ IL-2+ and CD4+ IL2+ T cells were not significantly altered by C-RT compared to RT alone. Finally, with both regimens, NK cells and B-cell numbers showed a more rapid recovery than T-cell numbers. CONCLUSION: Administration of concurrent cisplatinum to radiation may synergistically increase cytotoxic effects of radiation on tumor cells but does not alter the magnitude and the characteristics of radiation-induced immunosuppression.

Effects of irradiation on the expression of major histocompatibility complex class I antigen and adhesion costimulation molecules ICAM-1 in human cervical cancer.

PURPOSE: We initiated studies to analyze the effects of high doses of gamma irradiation on the surface antigen expression of MHC Class I, Class II, and ICAM-1 on human cervical carcinoma cell lines. METHODS AND MATERIALS: The expression of surface antigens (MHC Class I, Class II, and ICAM-1) was evaluated by FACS analysis on two cervical cell lines at different time points, following their exposure to high doses of gamma irradiation (i.e., 25.00, 50.00, and 100.00 Gy). RESULTS: The CaSki and SiHa cervical cancer cells we analyzed in this study expressed variable levels of MHC Class I and ICAM-1 antigens, while Class II surface antigens were not detectable. Whereas irradiation doses of 25.00 Gy were not sufficient to totally block cell replication in both cell lines, exposure to 50.00 or 100.00 Gy was able to completely inhibit cell replication. Range doses from 25.00 to 100.00 Gy significantly and consistently increased the expression of all surface antigens present on the cells prior to irradiation but were unable to induce neoexpression of antigens previously not expressed by these cells (i.e., MHC Class II). Importantly, such upregulation was shown to be dose dependent, with higher radiation doses associated with increased antigen expression. Moreover, when the kinetic of this upregulation was studied after 2 and 6 days after irradiation, it was shown to be persistent and lasted until all the cells died. CONCLUSIONS: These findings may partially explain the increased immunogenicity of tumor cells following irradiation and may suggest enhanced immune recognition in tumor tissue in patients receiving radiation therapy.

Down-regulation of the human c-fos and c-myc proto-oncogene promoters by adeno-associated virus Rep78.

Adeno-associated virus (AAV) is a non-pathogenic human parvovirus which has anti-tumor and anti-proliferation properties in tissue culture and animal studies. Furthermore, AAV infection is negatively associated with human cervical cancer. C-myc has been implicated in cervical cancer, and c-fos is involved in signal transduction initiation of cell growth. To study the potential regulation of these two prominent human proto-oncogenes by AAV, the expression of three marker coding sequences ligated 3' of the proto-oncogene promoters were observed. Demonstrated here, the AAV Rep78 gene product was able to down-regulate the human c-fos and c-myc proto-oncogene promoters in all three assay systems. These interactions may partially explain AAVs anti-proliferation properties.

The regulatory rep protein of adeno-associated virus binds to sequences within the c-H-ras promoter.

The large rep gene products (rep68 and rep78) of adeno-associated virus (AAV) are pleiotropic effector proteins which not only play a critical role in AAV DNA replication and in the trans-regulation of AAV promotor elements, but are also known for their onco-suppressive functions. We have previously demonstrated that the large AAV rep protein will strongly inhibit expression from the c-H-ras promoter, but not the murine osteosarcoma virus long terminal repeat (MSV-LTR) promoter. To investigate the possibility that rep may physically bind to these promoter sequences, specifically to GCTC motifs, we conducted electrophoretic mobility shift assays (EMSA) with a maltose binding protein-rep chimeric protein, MBP-rep68 delta, and synthetic double stranded DNA substrates of sequences selected from the c-H-ras and MSV-LTR promoters, as well as with the AAV TR. We find that MPB-rep68 delta bound the AAV TR DNA sequence (three motifs) most strongly, followed by the selected c-H-ras DNA sequence (two noninterfering motifs), and most poorly to the MSV-LTR DNA (one motif). These data are consistent with our previous study and suggest a direct mechanism of action for AAV rep inhibition of the c-H-ras promoter. Furthermore, the results suggest that the number of GCTC motifs, when closely associated, affect the affinity of rep binding. Finally, we find that MBP-rep68 delta also binds to the c-H-ras oligomer substrates which have secondary hairpin structures.