Saraswathy Seetharam

Combined gene therapy and ionizing radiation is a novel approach to treat human esophageal adenocarcinoma

BackgroundThe ability to infect tumor cells limits the antitumor effects of gene therapy. The addition of radiotherapy to treatment with Ad.Egr.TNF.11D, a replication-deficient adenovirus containing a radiation-inducible promoter, early growth response-1, and the tumor necrosis factor-α (TNFα) complementary DNA may enhance the therapeutic ratio.MethodsSeg-1 human esophageal adenocarcinoma cells were treated with Ad.Egr.TNF.11D with or without radiation. TNFα levels were quantified with enzyme-linked immunosorbent assay. Athymic nude mice bearing Seg-1 tumors were randomized to buffer, ionizing radiation, Ad.Egr.TNF.11D, and combination therapy. Tumor growth delay was used to compare treatment regimens. TNFα levels were measured in tumor homogenates and plasma.ResultsSeg-1 cells treated with Ad.Egr.TNF-11D and ionizing radiation demonstrated increased TNFα levels at 72 hours compared with cells exposed to vector alone (124±0 pg/mL vs. 31.11±22 pg/mL;P=.008). In vivo, Ad.Egr.TNF.11D-treated tumors expressed low TNFα levels (151.5 ±107.11 pg/mg protein) compared with tumors receiving combined treatment (793.92±489.13 pg/mg protein;P=.067). Increased TNFα levels were associated with increased tumor growth delay after combined treatment (P<.05).ConclusionsRadiotherapy enables focal stimulation of TNFα expression in Ad.Egr.TNF.11D-infected cells and thus improves local tumor control.

Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts

In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in Epstein-Barr virus transformed lymphoblastoid cell lines from a patient, XP12BE, with xeroderma pigmentosum (XP), group A, and a normal control. XP is a skin-cancer-prone disorder with UV hypersensitivity and defective DNA repair. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of E. coli. An earlier report on this data [Seetharam et al., (1990) J. Mol. Biol., 212, 433] indicated lower survival and higher mutation frequency with the UV-treated plasmid passed through the XP12Be(EBV) line. In the present report, sequence analysis of 198 mutant plasmids revealed a predominance of G:C----A:T transitions with both lymphoblastoid cell lines. This finding is consistent with the bias of polymerases toward insertion of an adenine opposite non-coding photoproducts (dinucleotides or other lesions). Transversion mutagenesis, non-adjacent double mutations, and triple-base mutations may involve other mechanisms. These results were compared to similar data from a fibroblast line from the same patient [Bredberg et al., (1986) Proc. Natl. Acad. Sci. (U.S.A.), 83, 8273]. The frequency of G:C----A:T transitions was higher, and there were fewer plasmids with multiple-base substitutions and with transversion mutations with both XP lymphoblasts and fibroblasts than with the normal lymphoblasts and fibroblasts. There were no significant differences in classes or types of mutations in the UV-treated plasmid replicated in the XP lymphoblasts and the XP fibroblasts. This suggests that the major features of UV mutagenesis in different cell types from the same individual are similar.

Tumor production of angiostatin is enhanced after exposure to TNF-α

Infection of tumors with an adenoviral vector expressing a chimeric gene composed of the CArG elements of the Egr‐1 promoter and a cDNA encoding TNF‐α (Ad.Egr‐TNF) has previously been shown to result in the production of high intratumoral levels of TNF‐α and thereby tumor regression. The antitumor effects of TNF‐α were ascribed to vascular thrombosis. We and others, have reported that inhibition of tumor vessel thrombosis using anticoagulation therapy does not abrogate the antitumor effects after TNF‐α treatment. To investigate the potential antiangiogenic effects of TNF‐α, we studied the generation of angiostatin after intratumoral injection of Ad.Egr‐TNF. We report an increase in plasma angiostatin levels both during and after treatment with Ad.Egr‐TNF that parallel tumor regression. We also report that TNF‐α enhances angiostatin production by inducing the activity of plasminogen activator and the release of MMP‐9 by tumor cells. These studies support a model in which the antiangiogenic effects of TNF‐α on the tumor microvasculature are mediated by generation of angiostatin.

Mutational hotspot variability in an ultraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts

The mutagenesis shuttle vector, pZ189, was treated with ultraviolet (u.v.) radiation in vitro and passed through a DNA repair-deficient lymphoblastoid cell line derived from a patient with xeroderma pigmentosum complementation group A (XP-A) (XP12BE(EBV)) and a DNA repair-proficient lymphoblastoid cell line (GM606(EBV)). After u.v. treatment, plasmid survival was lower and mutation frequency higher with the XP-A cells mirroring the survival and mutagenesis of the host cells. The nature of the mutations in the suppressor tRNA marker gene was determined by direct sequence analysis. The G.C to A.T transition was the dominant (85%) base substitution mutation with the XP lymphoblasts and was the major (56%) base substitution mutation with the repair-proficient lymphoblasts. We found a G.C to A.T transition mutational hotspot with the XP lymphoblasts not seen in our previous experiments with fibroblasts from the same patient. Comparison of the data presented here with our results with DNA repair-deficient and DNA repair-proficient fibroblasts suggests that hotspot variability is not due to genetic polymorphism or repair capacity of the cells. Instead it appears that cellular factors can influence the probability of mutagenesis of modified DNA at particular sites.

Angiostatin potentiates cyclophosphamide treatment of metastatic disease

Abstract Purpose. We examined the interaction between cyclophosphamide (CPA) and angiostatin (AS) on the growth of primary Lewis lung carcinoma (LLC) tumors and on the development of LLC pulmonary metastases. We studied the effects of AS and CPA on the stages of angiogenesis employing in vitro assays. Methods. Primary tumor growth and pulmonary metastases were measured to evaluate the effects of treatment with AS alone, CPA alone or the combination of CPA and AS. We examined the effects of CPA plus AS on endothelial cell (HUVEC) survival, migration and tube formation. Results. Combined treatment with CPA and AS did not significantly affect primary tumor growth when compared with CPA treatment alone. However, a significant decrease in the number of pulmonary metastases was observed following CPA plus AS treatment when compared with CPA treatment alone (P<0.001). AS did not enhance CPA-mediated HUVEC cytotoxicity, and CPA failed to enhance AS-mediated inhibition of migration. However, tube formation was inhibited following combined treatment with CPA and AS when compared with either treatment alone. Conclusions. AS enhanced the antimetastatic effects of CPA without significantly influencing the effects of CPA on primary tumor growth. CPA plus AS inhibited tube formation, suggesting that interrupting specific steps in the angiogenesis process might be an effective approach to the treatment of subclinical distant metastases.

Modulation of ultraviolet light mutational hotspots by cellular stress

Stressful treatments of cells provoke broad, transient, changes in cellular physiology and gene expression. In addition to these effects, DNA-damaging agents often induce permanent change in the form of mutations. Mutational patterns in target genes typically show hotspots and coldspots, the molecular basis of which appears to lie in the sequence context of the particular site. We determined the mutational pattern in an ultraviolet light-modified (in vitro) marker gene in a shuttle vector passaged through repair deficient (xeroderma pigmentosum) cells and compared it with patterns obtained from cells exposed to stress imposed by a DNA-damaging agent or a calcium ionophore. We found that the mutational hotspot pattern was altered by both stress treatments. We conclude that the cellular environment can influence the probability of mutagenesis at specific sites and propose that some of these effects on mutagenesis are mediated by alterations in cellular calcium levels.