Detection of repair activity during the DNA damage-induced G2 delay in human cancer cells.

All eukaryotic cells manifest cell cycle delay after exposure to DNA damaging agents. It has been proposed that such cell cycle checkpoints may allow DNA repair but direct evidence of such activity during the radiation-induced G2 delay has been lacking. We report here that cells arrested in G2 by radiation (2-3 Gy) and etoposide incorporate bromodeoxyuridine (BrdU) at discrete foci in the nucleus. We detected G2 cells with CENP-F, a nuclear protein maximally expressed in G2. Caffeine and okadaic acid, both established radiosensitizers, inhibit the incorporation of BrdU in G2 cells. Radioresistant HT29 and OVCAR cells demonstrate BrdU foci formation more frequently during the G2 delay when compared to the more radiosensitive A2780 cell line. The repair foci formed during G2 may be followed through mitosis and observed in daughter cells in G1. Taken together, these observations are consistent with the detection of DNA repair activity during the radiation-induced G2 delay after relatively low doses of radiation.

Use of complementary health practices by prostate carcinoma patients undergoing radiation therapy.

BACKGROUND: There has been increasing interest in complementary health practices among patients, popular media, and even institutional health care providers. However, there is still surprisingly little information on the use of alternative medicine by patients undergoing treatment for prostate carcinoma. METHODS: A prospective study of 50 consecutive patients undergoing radiation treatment for prostate carcinoma at a tertiary care referral center was conducted. Patients were surveyed after the initiation of radiation therapy whether they had used complementary health practices at any time. RESULTS: The authors found that a surprisingly high proportion of patients (37%) relied on complementary health practices not prescribed by physicians. In contrast, according to a separate survey of the treating physicians, the physicians believed that on average only 4% of their patients resorted to such practices. The use of complementary health practices usually continued even after the initiation of definitive treatment for prostate carcinoma. Patients who used complementary health practices tended to have higher levels of education and income, whereas there were no differences in age, religion, perception of health status, stage of prostate carcinoma, or prostate specific antigen level. Herbal remedies were the most frequently utilized, by 60% of those using complementary health practices, followed by old-time remedies (47%), high dose vitamins (41%), chiropractic/massage therapy and relaxation techniques (18% each), and special diets (12%). CONCLUSIONS: Patients undergoing radiation therapy for prostate carcinoma frequently rely on complementary health practices not prescribed by their treating physicians. Patients who do so tend to have higher education and income levels and continue their complementary practices during the conventional treatment. As the health implications of these practices are unclear, further research is clearly needed.

p34(Cdc2) kinase activity is excluded from the nucleus during the radiation-induced G(2) arrest in HeLa cells.

The progression of cells from G(2) into mitosis is blocked by exposure to DNA-damaging agents such as ionizing radiation. This G(2) delay is associated with reduced cyclin B1-specific associated histone H1 kinase activity, increased inhibitory phosphorylation of p34(Cdc2), and depressed cyclin B1 levels in HeLa cells. Induction of cyclin B1 or expression of Cdc2AF, a mutant p34(Cdc2) that lacks the sites of inhibitory phosphorylation, only partially reverses the radiation-associated G(2) delay, although both maneuvers rapidly result in increased histone H1 kinase activity. To account for the persistent G(2) delay in the face of active p34(Cdc2) kinase, we determined the location of the kinase activity. Although p34(Cdc2) was active in the cytoplasm, the nuclear p34(Cdc2) was inactive. Irradiation led to nuclear accumulation of the inactive tyrosine-phosphorylated form of p34(Cdc2), whereas the active form was seen in the cytoplasm. At later times when cells had resumed cell cycle progression, nuclear kinase activity was detectable. These results give evidence of segregation of cytoplasmic and nuclear kinase activity after DNA damage that has the effect of enhancing checkpoint control. Shielding the nucleus from the potentially deleterious effects of kinase activity after DNA damage may help irradiated human cancer cells respond to irradiation.

Oral cavity and oropharyngeal tumors in human immunodeficiency virus-positive patients: acute response to radiation therapy.

BACKGROUND: The survival of patients with human immunodeficiency virus (HIV) has improved considerably with modern medical management. However, there remains surprisingly little information on treating head and neck neoplasms in HIV-positive patients. OBJECTIVE: To report our recent experience treating oral cavity and oropharyngeal tumors in HIV-positive patients. DESIGN AND PATIENTS: Retrospective analysis of a cohort of 8 HIV-positive patients with Kaposi sarcoma (KS), lymphoma, or squamous carcinoma of the oral cavity or oropharynx who were consecutively treated during a single year with radiation therapy at a tertiary care referral center. Length of follow-up was at least 2 years (mean, 2.5 years). RESULTS: All patients had partial and complete responses to treatment lasting until the last follow-up. However, we found that treatment was considerably better tolerated by patients with non-KS tumors, with fewer acute reactions and significantly less weight loss, despite larger treatment volumes and higher radiation doses, compared with patients with KS. Patients with non-KS tumors received a mean radiation dose of 62.6 Gy to 2636 cm3, yet lost only a mean of 0.1 kg in weight, whereas patients with KS were treated with a mean radiation dose of 19 Gy to a mean volume of 568 cm3, but lost a mean of 5.8 kg during treatment (P = .005) and on average sustained an additional grade of severity on a standard scale of mucosal reaction (P = .01). CONCLUSIONS: Oral cavity and oropharyngeal tumors in HIV-positive patients respond to radiation therapy, but there is a marked difference in the degree of acute reactions to treatment between patients with and without KS. Infection with HIV is not a contraindication when aggressive radiation therapy is needed in select patients.

The utility of serial complete blood count monitoring in patients receiving radiation therapy for localized prostate cancer.

PURPOSE: It is standard practice in our department to monitor weekly complete blood counts (CBCs) in patients receiving definitive radiation therapy for prostate cancer. The clinical utility and cost effectiveness of this practice has not been analyzed. METHODS AND MATERIALS: The charts of all prostate cancer patients treated with radiation therapy between January 1994 and July 1996 at the Veterans Administration Hospital, Philadelphia, PA were reviewed. CBC values were available for 89 patients. Patients received a median dose of 68 Gy using a four-field box technique and megavoltage photons. Whole-pelvic radiotherapy followed by a conedown to the prostate was administered to 29 patients. Fifty-nine patients received radiation to the prostate alone or prostate and seminal vesicles. Fifty-seven patients received concurrent hormonal therapy which included luteinizing hormone-releasing hormone (LHRH) agonist, antiandrogens, or both. RESULTS: No patient experienced a drop in their hemoglobin, white blood cells (WBCs), or platelets below critical nadirs (defined as WBC < 2 counts x 1000/mm(3), hemoglobin < 8 g/dl, platelet < 50 counts x 1000/mm(3) 2 in WBCs. In the urban area surrounding the Philadelphia Veterans Administration Medical Center, the cost of obtaining a CBC is approximately $30. However, if staff time is considered, the cost of obtaining a weekly CBC during prostate cancer radiotherapy approached $400 per patient. CONCLUSION: These results suggest that weekly monitoring of CBCs in prostate cancer patients undergoing definitive radiotherapy may not be necessary. We recommend a baseline CBC be performed, and if normal, no other monitoring unless clinically indicated. This strategy would result in a cost savings approaching $30,000 per 100 treated patients. Further research on the cost effectiveness and utility of serial blood tests in patients receiving partial body radiation therapy is needed.

Conversion of a radioresistant phenotype to a more sensitive one by disabling erbB receptor signaling in human cancer cells.

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by gamma-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to gamma-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.

HIV-1 Vpr interacts with a human 34-kDa mov34 homologue, a cellular factor linked to the G2/M phase transition of the mammalian cell cycle.

Several important and possibly interrelated functions have been identified for the HIV-1 accessory gene product Vpr. These include import of the HIV reverse transcription complex into the nucleus of nondividing cells, cellular differentiation including cell cycle arrest at the G2/M phase border, immune suppression, and enhancement of virus replication. We have cloned a candidate Vpr ligand, termed human Vpr interacting protein (hVIP/MOV34), by using a yeast two-hybrid assay. This gene is homologous to a simultaneously identified 34-kDa human mov34 homologue. The MOV34 family includes proteins that function as transcriptional and proteolytic regulators of cell growth and differentiation. We demonstrate direct interactions between the putative ligand hVIP/MOV34 and Vpr in vitro and in vivo. hVIP/MOV34 localizes to the nucleus and appears to function as a component of the cell cycle cascade. We observe an association between the induction of cell cycle arrest at the G2/M phase border by Vpr and a change in the subcellular localization of hVIP/MOV34 from a nuclear to a perinuclear localization. This was further associated with the inhibition of maturation promoting factor-associated histone H1 kinase activity. We conclude that hVIP/MOV34 is involved in the regulation of the cell cycle and a likely cellular cofactor for HIV-1 Vpr.

The molecular regulation of apoptosis and implications for radiation oncology.

One of the major goals of cancer research is to identify and understand the causes of cellular proliferation. The role of cell death, or lack thereof, in carcinogenesis, tumour growth, metastatic spread and response to treatment has been largely overlooked even though the morphology of apoptosis (programmed cell death) was clearly described over 20 years ago, and its importance in cancer speculated on at that time. Over the last 5 years, however, an explosion of research has focused on delineating the molecular components of the apoptotic pathways and examining the role of apoptosis in a tumour's growth and response to treatment. This review highlights the aspects of apoptosis most relevant to radiation oncologists and radiobiologists. The apoptotic pathways will be described, with attention to the stimuli that initiate apoptosis, the oncogenes and tumour suppressor genes that mediate apoptosis, and the effector enzymes (proteases and endonucleases) responsible for the execution of apoptosis. In addition, we review the effect of classically described radiobiology cell survival parameters-cell cycle stage, dose rate, linear energy transfer, oxygen, total dose, and fractionation-on radiation induced apoptosis.

Cyclin B1 availability is a rate-limiting component of the radiation-induced G2 delay in HeLa cells.

Irradiation of tumor cells results in a G2 delay, which has been postulated to allow DNA repair and cell survival. The G2 delay after irradiation is marked in HeLa and other cells by delayed expression of cyclin B1. To test whether this depression of cyclin B1 contributes to the G2 delay, we induced cyclin B1 expression in irradiated HeLa cells using a dexamethasone-inducible promoter. Induction of cyclin B1 after radiation abrogated the G2 delay by approximately doubling the rate at which the cells reentered mitosis, whereas dexamethasone itself had no effect. However, overexpression of cyclin B1 did not eliminate the G2 delay in irradiated cells. In unirradiated cells, overexpression of cyclin B1 had no effect on cell cycle progression. Confirmation that reduction of cyclin B1 levels would prolong G2 was provided using antisense oligonucleotides to cyclin B1. These results demonstrate that cyclin B1 levels control the length of the G2 delay following irradiation in HeLa cells but do not exclude additional mechanisms controlling the mitotic delay after irradiation.

Potential molecular targets for manipulating the radiation response.

Recent advances in our 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. Specific targets which, if inactivated, might increase radiosensitivity include Ras, which has been implicated in the radioresistant phenotype, and components of DNA-dependent protein kinase or other molecules involved in the recognition or repair of DNA damage. In some tumors, apoptosis is an important mode of cell death following radiation, so agents that promote this may prove useful therapeutically. Conversely, side effects may result from radiation-induced apoptosis of normal tissues: for example, pneumonitis following the destruction of endothelial cells in the pulmonary vasculature. Therefore, decreasing apoptosis in these tissues may reduce late effects. It may also be possible to prevent late effects such as fibrosis by blocking the induction of certain genes such as transforming growth factor beta. Cell-cycle regulation is another area that could be manipulated to increase radiosensitivity. There is evidence that the G2 delay following radiation is important in protecting cells from death. Abolition of this delay may increase radiosensitivity, especially in cells with mutant p53 that have lost the G1 checkpoint.

Locally advanced renal cell carcinoma: low complication rate and efficacy of postnephrectomy radiation therapy planned with CT.

PURPOSE: To determine the usefulness of computed tomography (CT) in planning radiation therapy for locally advanced renal cell carcinoma after nephrectomy. MATERIALS AND METHODS: The authors retrospectively analyzed the treatment records and follow-up status of 12 consecutive patients who underwent radical nephrectomy and postoperative radiation therapy for locally advanced renal cell carcinoma. Records' of 12 consecutive patients who underwent only radical nephrectomy were also analyzed. RESULTS: None of the patients who received radiation therapy after nephrectomy had local-regional recurrence, despite disease at the surgical margins in six patients; the actuarial disease-free survival at 5 years was 75%. In contrast, the 5-year actuarial local failure rate in the surgery-only group was 30% (significant difference at P < .01) and the disease-free survival rate was 62% (mean and median follow-up was 4.6 and 5.1 years, respectively). CONCLUSION: With CT, radiation can be delivered to the renal bed safely and without undue morbidity. Given the lack of chronic complications associated with the side effects of radiation therapy and uniform local control of cancer in these patients, the role of radiation therapy in patients at high risk for local failure may be reconsidered.

The impact of perioperative factors on subsequent intelligence quotient deficits in children treated for medulloblastoma/posterior fossa primitive neuroectodermal tumors.

BACKGROUND: Despite success in treating children with medulloblastoma/posterior fossa primitive neuroectodermal tumor (PF PNET), some children survive with significant neurocognitive sequelae. This study was performed to understand better the significance of perioperative factors on subsequent full scale intelligence quotient (FSIQ) deterioration in these children. METHODS: Twenty-eight children who underwent prospective and serial neurocognitive testing were studied. All children underwent surgery followed by radiotherapy with or without chemotherapy between 1983 and 1987 for medulloblastoma/PF PNET and were disease free when this study was conducted. IQ testing was performed before surgery and after the completion of radiation therapy. The clinical courses of the patients were correlated with changes in the corresponding intelligence quotients of each child. Factors correlating with neurocognitive declines were examined by chi-square or Fisher exact test analysis. Differences in mean IQs were examined by the t test. Factors found to be significant were analyzed by exact logistic regression analysis. RESULTS: The presence of adverse factors such as neurologic deficits, meningitis, or shunt infections, or the need for repeat surgery was correlated significantly with IQ deficits after treatment. Of the subset of children with one or more of these factors, 13 of 16 (81%) sustained decreases in FSIQ; 7 of 16 (43.8%) had decreases of 20 points of more. In contrast, only 3 of 12 (25%) of the children without the factors sustained FSIQ decreases, and no child sustained a decrease of more than 13 points. The mean FSIQ change after treatment in the group with factors was -15.7 (95% confidence interval [CI]: -24.0, -8.4), and the median was -18. The mean FSIQ change in the group without factors was 4.8 (95% CI: -0.5, 10.1), and the median was 5. The difference in mean FSIQ change between the two groups was significant (P < 0.0001). On univariate analysis, both the presence of adverse factors and an age less than six years correlated with neurocognitive deficit. On regression analysis, only the presence of adverse factors was significant (odds ratio 11.53; 95% CI, 1.65-116.58; P = 0.009), whereas age was not (P = 0.27). CONCLUSIONS: Perioperative events or complications may account for some of the neurocognitive deterioration seen in these children after treatment, especially in the very young. The occurrence of these factors is associated with a significantly greater risk of IQ deterioration. Studies of the neurocognitive effects of treatment for children with medulloblastoma/PF PNET should include an analysis of these postoperative factors.

Anatomy of the celiac axis and superior mesenteric artery and its significance in radiation therapy.

PURPOSE: In the radiation therapy of upper gastrointestinal malignancies, treatment of lymph nodes in the region of the celiac axis and superior mesenteric axis is often mandated. This study was undertaken to determine the relationship of the celiac axis and superior mesenteric arteries to the vertebral bodies--the radiographically visualized reference structures during simulation. METHODS AND MATERIALS: Twenty-three celiac angiograms and 24 superior mesenteric angiograms performed preoperatively in 24 patients treated at the University of Pennsylvania from 1984 to 1989 for pancreatic carcinoma were examined. The location of the origin of the celiac and superior mesenteric arteries was determined in each case. RESULTS: In 48% of the celiac angiograms, the celiac axis arose from the aorta high at the pedicle of the T-12 vertebral body, contrary to the common belief that the celiac axis arises near the T12-L1 interspace. The superior mesenteric artery arose at the level of L-1 in 83% of the 24 angiograms and below the pedicle of L-1 in 5 (21%). However, none arose below the L1-2 interspace. CONCLUSION: The variability demonstrated in the levels from which these vessels arise strongly suggests individualized treatment planning, including angiographic, CT or MRI data should be performed if tight margins are used. These studies would additionally optimize treatment of the tumor bed. Consideration for dose at field edges (i.e., "buildup") and day to day variation in set-up is required in determining the field borders. Treatment volumes tightly encompassing T12 and L1 could risk undertreating regional lymph nodes associated with these vessels.