Downregulation of the Rho GTPase pathway abrogates resistance to ionizing radiation in wild-type p53 glioblastoma by suppressing DNA repair mechanisms.

Glioblastoma (GBM), the most common aggressive brain tumor, is characterized by rapid cellular infiltration and is routinely treated with ionizing radiation (IR), but therapeutic resistance inevitably recurs. The actin cytoskeleton of glioblastoma cells provides their high invasiveness, but it remains unclear whether Rho GTPases modulate DNA damage repair and therapeutic sensitivity. Here, we irradiated glioblastoma cells with different p53 status and explored the effects of Rho pathway inhibition to elucidate how actin cytoskeleton disruption affects the DNA damage response and repair pathways. p53-wild-type and p53-mutant cells were subjected to Rho GTPase pathway modulation by treatment with C3 toxin; knockdown of mDia-1, PFN1 and MYPT1; or treatment with F-actin polymerization inhibitors. Rho inhibition increased the sensitivity of glioma cells to IR by increasing the number of DNA double-strand breaks and delaying DNA repair by nonhomologous end-joining in p53-wild-type cells. p53 knockdown reversed this phenotype by reducing p21 expression and Rho signaling activity, whereas reactivation of p53 in p53-mutant cells by treatment with PRIMA-1 reversed these effects. The interdependence between p53 and Rho is based on nuclear p53 translocation facilitated by G-actin and enhanced by IR. Isolated IR-resistant p53-wild-type cells showed an altered morphology and increased stress fiber formation: inhibition of Rho or actin polymerization decreased cell viability in a p53-dependent manner and reversed the resistance phenotype. p53 silencing reversed the Rho inhibition-induced sensitization of IR-resistant cells. Rho inhibition also impaired the repair of IR-damaged DNA in 3D spheroid models. Rho GTPase activity and actin cytoskeleton dynamics are sensitive targets for the reversal of acquired resistance in GBM tumors with wild-type p53.

Radiation enhances the invasive potential of primary glioblastoma cells via activation of the Rho signaling pathway.

Glioblastoma multiforme (GBM) is among the most treatment-refractory of all human tumors. Radiation is effective at prolonging survival of GBM patients; however, the vast majority of GBM patients demonstrate progression at or near the site of original treatment. We have identified primary GBM cell lines that demonstrate increased invasive potential upon radiation exposure. As this represents a novel mechanism by which radiation-treated GBMs can fail therapy, we further investigated the identity of downstream signaling molecules that enhance the invasive phenotype of irradiated GBMs. Matrigel matrices were used to compare the extent of invasion of irradiated vs. non-irradiated GBM cell lines UN3 and GM2. The in vitro invasive potential of these irradiated cells were characterized in the presence of both pharmacologic and dominant negative inhibitors of extracellular matrix and cell signaling molecules including MMP, uPA, IGFR, EGFR, PI-3K, AKT, and Rho kinase. The effect of radiation on the expression of these signaling molecules was determined with Western blot assays. Ultimately, the in vitro tumor invasion results were confirmed using an in vivo 9L GBM model in rats. Using the primary GBM cell lines UN3 and GM2, we found that radiation enhances the invasive potential of these cells via activation of EGFR and IGFR1. Our findings suggest that activation of Rho signaling via PI-3K is required for radiation-induced invasion, although not required for invasion under physiologic conditions. This report clearly demonstrates that radiation-mediated invasion is fundamentally distinct from invasion under normal cellular physiology and identifies potential therapeutic targets to overcome this phenomenon.

Biologic factors and response to radiotherapy in carcinoma of the cervix.

Ionizing radiation has been used to treat cancers for a century. However, radioresistance remains a major problem in the clinic. Recent advances in the understanding of the molecular events that occur following ionizing radiation leading to DNA damage and repair, apoptosis, and cell cycle arrests suggest new ways in which the radiation response might be manipulated. Seventy-eight cases of carcinoma of the cervix of the same stage (II A and B) were analyzed retrospectively. All patients were treated with radiotherapy (RT) with a dose varying from 35 Gy to 50 Gy with 200 cGy per fraction. Subsequent to the completion of radiotherapy, all patients underwent surgery 4-6 weeks later. On histological examination of the surgical specimens, 51% of the cases (40) showed a complete response to therapy with no viable tumor cells. 49% of cases (38) had residual tumors ranging from a small focus to lesions extending through more than half the thickness of the cervical wall. p53 (mutant), bcl-2, p21 and bax proteins were studied on the paraffin sections of the biopsies (pretreatment) of those patients who failed to respond to RT and compared to similar studies on biopsies of patients who had a complete response to RT. In addition, the minichromosome maintenance (MCM) 2 proliferative marker was also done on all cases. Expression of all proteins was done using immunohistochemsitry. In the radioresistant cases, 15% (six cases) showed positivity for bcl-2 and p21, respectively, and 34% (13 cases) showed mutant p53. None of the radiosensitive tumors were positive for the above proteins. 75% of the radiosensitive tumors (30 cases) were positive for the bax antibody, whereas 81% of the radioresistant tumors (31 cases) were negative for bax. The MCM2 proliferative marker was positive in > 80% of cells in 81.5% of radioresistant tumors (31 cases) as compared to < 40% of cells that were positive in 70% of radiosensitive tumors (28 cases). The P-value for the biological markers was calculated using the chi-squared test, and was highly significant (P < 0.01) for all the parameters tested. However, there was no statistical significance by univariate analysis when the dose of radiation was analyzed with respect to the markers and the histological response. There was also no correlation between the radiation response and timing of surgery. The above data strongly suggest that bax, along with proliferative markers, could play a role in determining which tumors are likely to respond to radiation therapy. The presence of bcl-2, p21 and p53 could also be related to radioresistance of the tumors.