Gregory J. Duigou

Ionizing radiation modulates vascular endothelial growth factor (VEGF) expression through multiple mitogen activated protein kinase dependent pathways.

We investigated the role of radiation-induced mitogen activated protein kinase (MAPK) pathway activity in the regulation of proliferation, cell survival and vascular endothelial growth factor (VEGF) production in primary astrocytes and in T9 and RT2 glioblastoma cells derived from Fisher 344 rats. In these cells, ionizing radiation (2 Gy) caused activation of the MAPK pathway which was blocked by specific inhibitor drugs. Blunting of radiation-induced MAPK activity weakly enhanced radiation-induced apoptosis 24 h after exposure in RT2 cells. Furthermore, blunting of MAPK activation weakly enhanced the ability of radiation to reduce RT2 cell growth in clonogenic growth assays. These findings argue that inhibition of MAPK signaling reduces proliferation and enhances cell killing by ionizing radiation in transformed astrocytes. Proliferation and survival of cancer cells has been linked in vivo to enhanced expression of angiogenic growth factors. Recently we demonstrated that the gene product of a novel rodent radiation-responsive gene, progression elevated gene 3 (PEG-3), could enhance vascular endothelial growth factor (VEGF) promoter activity in rodent fibroblasts, leading to increased VEGF protein levels and tumorigenic behavior in vivo. Thus PEG-3 and VEGF expression could be expected to directly correlate with the oncogenic potential of transformed cells. RT2 cells expressed more PEG-3 and VEGF protein than T9 cells, and were more tumorigenic in vivo than T9 cells. Radiation activated the PEG-3 promoter via MAPK signaling and ectopic over-expression of PEG-3 enhanced both basal MAPK activity and basal VEGF promoter activity. Basal MAPK activity partially correlated with basal VEGF promoter activity and VEGF protein levels in primary astrocytes, T9 and RT2 cells. Radiation increased the activity of the VEGF promoter and VEGF protein levels in primary astrocytes, T9 and RT2 cells which were dependent upon MAPK function. Furthermore, inhibition of AP-1 transcription factor signaling by dominant negative c-Jun (TAM67) also significantly reduced basal, and to a lesser extent radiation-induced, VEGF promoter function in RT2 cells. Collectively, our data demonstrate that radiation-induced MAPK signaling can both protect cells from radiation-induced cell death as well as enhance protein levels of pro-angiogenic factors such as VEGF. Enhanced VEGF expression in RT2 cells may be mediated via MAPK and JNK pathway signaling which converges upon the AP-1 transcription factor complex.

Decreased Replication Ability of E1-Deleted Adenoviruses Correlates with Increased Brain Tumor Malignancy

E1 region replacement adenoviruses are replication defective and are propagated in cells providing adenovirus E1A and E1B proteins. Although they are being developed for antitumor therapies, the proliferative behaviors of these viruses in normal brain tissues or in brain tumors are unknown. To address this, freshly cultured cells from normal human brain and common brain tumors (astrocytomas and meningiomas) were infected using wild-type species C adenoviruses and adenoviruses missing E1A (H5dl312) or E1A plus E1B (H5dl434). Viral DNA replication, late viral protein expression, and production of infectious progeny were characterized. Wild-type adenoviruses grew efficiently in normal brain and brain tumor cells. In comparison, E1-deleted adenovirus DNA replication was delayed and lower in cells derived from normal brain tissues, meningiomas, and low-grade astrocytomas. However, in contrast, E1-deleted adenovirus DNA replication did not occur or was extremely low in cells derived from malignancy grade III and IV astrocytic tumors. Because wild-type adenoviruses infected and replicated in all cells, the malignancy grade-based differential E1-deleted adenovirus DNA replication was not explained by differential virus uptake. Infectious H5dl312 and H5dl434 production correlated with viral DNA replication. Compared with a 5-day average for wild-type infections, advanced cytopathology was noted approximately 4 weeks after H5dl312 or H5dl434 infection of meningioma, astrocytoma, and normal brain cells. Cytopathology was not observed after H5dl312 or H5dl434 infection of glioblastoma, anaplastic astrocytoma, and gliosarcoma cells. Because of this tumor grade-based differential growth, the E1-deleted adenoviruses may represent novel tools for studies of brain tumor malignancy.

Transcription analysis of TIMP-1 and NM23-H1 genes in glioma cell invasion.

PURPOSE To evaluate using transcription analysis the presence and importance of two genes: NM23-H1 and TIMP-1 on control of tumor cell invasion in diffuse astrocytomas (WHO II) and glioblastoma multiforme (WHO IV). METHOD Northern blot analysis of NM23-H1 and TIMP-1 was performed. Eight diffuse astrocytomas and 19 glioblastomas (WHO IV) were analyzed to determine if TIMP-1 and NM23-H1 were candidates to inhibition of tumor cell invasion quantitated RNA levels. The samples were collected directly from operating room. Total cellular RNA was extracted from frozen tissue samples using guanidinium-isothiocyanate and cesium chloride gradients. Total RNA (10 mg per sample) from tumor tissue were size fractionated through 1% agarose-formaldehyde gel and transferred to nylon filters and then hybridized to 32P-labeled DNA probes and placed for autoradiography. Levels of specific RNAs were determined by computer-assisted laser densitometry. Blot filters were sequentially hybridized to nm23 and TIMP-1 probes in addition to GAPDH, as a control. Statistical analyses were carried out according to t-test for equality of means. RESULTS NM23-H1 were detected in each sample, however it did not correlate with malignancy and invasiveness. On the other side TIMP-1 gene expression showed a clear correlation between low expression and invasiveness. CONCLUSION The data suggest that TIMP-1 is an inhibitor of high grade gliomas invasion. NM23-H1 was present in the entire gliomas sample, but it did not vary in diffuse astrocytomas and glioblastomas.

Suppression of the progression phenotype in somatic cell hybrids occurs in the absence of altered adenovirus type 5 gene expression

In the present study we have analyzed the genetic regulation of increased expression of transformation-associated traits, a process termed progression, in adenovirus type 5 (Ad5)-transformed secondary rat embryo cells. Somatic cell hybrids were formed between a highly progressed neomycin-resistant Ad5-transformed cloned cell line (E11-NMTneo) and an untransformed chloramphenicol-resistant rat embryo fibroblast cell line (CREFcap). Parental E11-NMTneo cells grew with high efficiency in agar, exhibited reduced 125I-epidermal growth factor (EGF) binding, and were tumorigenic in nude mice. Parental CREFcap cells exhibited phenotypes opposite to those of E11-NMTneo cells. A high proportion (84%) of the presumptive hybrid cell types obtained after fusion and genetic selection (G418 and chloramphenicol) displayed a flat morphological phenotype intermediate between CREFcap and E11-NMTneo cells, suggesting that a trans-dominant extinction phenomenon had occurred. Two hybrids with a round morphology (R), which still exhibited the progressed phenotype, and two hybrids with a flat morphology (F), which had lost the progressed phenotype, were chosen for detailed analysis. Both R hybrids grew efficiently in agar, exhibited low 125I-EGF binding, and were tumorigenic in nude mice, whereas both F hybrids grew poorly in agar, displayed increased 125I-EGF binding in comparison with E11-NMTneo and R hybrids, and were nontumorigenic in nude mice. An analysis of the viral DNA integration patterns and the rates of transcription, steady-state mRNA accumulation, and relative levels of the Ad5 E1A and E1B gene products revealed no differences among the parental and hybrid cells. These studies indicate that normal CREF cells may contain a suppressor gene(s) which can inhibit the expression of specific traits of the progression phenotype in Ad5-transformed cells and that this suppression is not associated with changes in the expression of Ad5 transforming genes.