Concurrent thyrolipomatosis and thymolipoma in a patient with myasthenia gravis: a case report and review of the literature.

We present a case of a man with a background of myasthenia gravis who presented with a neck lump, which was diagnosed as thyrolipomatosis in continuity with a very large thymolipoma. Following removal of these lesions, the patient's myaesthenic symptoms improved. While thymolipomas are often seen in the context of myasthenia gravis, thyrolipomatosis is a rare entity and to our knowledge the concurrent finding of both lesions with myasthenia gravis has never been reported. We highlight the important imaging features of both entities and the clinical importance of recognising them.

Ethmoidal metastasis as a first presentation of breast cancer: a case report.

Distant metastasis in advanced breast cancer is not uncommon; however, spread to the paranasal sinuses is extremely rare. We present a case of a woman who presented to our ophthalmology colleagues with worsening unilateral proptosis secondary to a tumour mass within her ethmoid sinuses. Biopsy of the ethmoid tumour showed adenocarcinoma of unknown origin. Whole-body positron emission computed tomography demonstrated a breast primary lesion. The patient was treated with palliative chemotherapy, and the patient remains well at this point. The importance of specialist head and neck radiological interpretation of imaging cannot be underestimated. Early tissue diagnosis is essential before ascribing patients with orbital symptoms to non-malignant process.

Consolidative proton therapy after chemotherapy for patients with Hodgkin lymphoma.

BACKGROUND: We investigated early outcomes for patients receiving chemotherapy followed by consolidative proton therapy (PT) for the treatment of Hodgkin lymphoma (HL). PATIENTS AND METHODS: From June 2008 through August 2015, 138 patients with HL enrolled on either IRB-approved outcomes tracking protocols or registry studies received consolidative PT. Patients were excluded due to relapsed or refractory disease. Involved-site radiotherapy field designs were used for all patients. Pediatric patients received a median dose of 21 Gy(RBE) [range 15-36 Gy(RBE)]; adult patients received a median dose of 30.6 Gy(RBE) [range, 20-45 Gy(RBE)]. Patients receiving PT were young (median age, 20 years; range 6-57). Overall, 42% were pediatric (≤18 years) and 93% were under the age of 40 years. Thirty-eight percent of patients were male and 62% female. Stage distribution included 73% with I/II and 27% with III/IV disease. Patients predominantly had mediastinal involvement (96%) and bulky disease (57%), whereas 37% had B symptoms. The median follow-up was 32 months (range, 5-92 months). RESULTS: The 3-year relapse-free survival rate was 92% for all patients; it was 96% for adults and 87% for pediatric patients (P = 0.18). When evaluated by positron emission tomography/computed tomography scan response at the end of chemotherapy, patients with a partial response had worse 3-year progression-free survival compared with other patients (78% versus 94%; P = 0.0034). No grade 3 radiation-related toxicities have occurred to date. CONCLUSION: Consolidative PT following standard chemotherapy in HL is primarily used in young patients with mediastinal and bulky disease. Early relapse-free survival rates are similar to those reported with photon radiation treatment, and no early grade 3 toxicities have been observed. Continued follow-up to assess late effects is critical.

Qualitative and quantitative differentiation of gases using ZnO thin film gas sensors and pattern recognition analysis.

In the present work we have grown highly textured, ultra-thin, nano-crystalline zinc oxide thin films using a metal organic chemical vapor deposition technique and addressed their selectivity towards hydrogen, carbon dioxide and methane gas sensing. Structural and microstructural characteristics of the synthesized films were investigated utilizing X-ray diffraction and electron microscopy techniques respectively. Using a dynamic flow gas sensing measurement set up, the sensing characteristics of these films were investigated as a function of gas concentration (10-1660 ppm) and operating temperature (250-380 °C). ZnO thin film sensing elements were found to be sensitive to all of these gases. Thus at a sensor operating temperature of ~300 °C, the response% of the ZnO thin films were ~68, 59, and 52% for hydrogen, carbon monoxide and methane gases respectively. The data matrices extracted from first Fourier transform analyses (FFT) of the conductance transients were used as input parameters in a linear unsupervised principal component analysis (PCA) pattern recognition technique. We have demonstrated that FFT combined with PCA is an excellent tool for the differentiation of these reducing gases.

Increasing sensitivity to radiotherapy and chemotherapy by using novel biological agents that alter the tumor microenvironment.

Sensitivity to radiation and chemotherapy can be influenced by factors extrinsic to the cancer cell. For example, severely hypoxic cells require 2-3 times the radiation dose as do well-oxygenated cells to achieve similar cell killing. Apart from the tumor cells, neighboring cells such as endothelial cells may influence radiosensitivity. Irradiation can lead to expression of molecules that may increase radio/chemoresistance, for example vascular endothelial growth factor (VEGF), a secreted protein that regulates angiogenesis, or hypoxia inducible factor-1 alpha (HIF-1 alpha), a master transcription factor that regulates gene expression in hypoxia. Hence, response to cytotoxic therapy may be improved by modulating the tumor microenvironment (TME). Several agents in clinical use may do this. Some of these target vasculature, either directly or indirectly by disrupting VEGF and/or HIF-1 signaling. Many of these agents have been shown to increase radio/chemosensitivity in preclinical models. A confounding factor in terms of radiosensitization is the variable effect of these drugs on tumor oxygenation. Some of these agents ablate the vasculature, thereby increasing hypoxia. Others may normalize tumor vasculature, leading to increased blood flow and oxygenation, thereby potentially increasing radiosensitivity and the delivery of chemotherapy. Inhibitors of EGFR signaling and the PI3K/Akt pathway can also cause similar vascular changes in preclinical models, perhaps by indirectly inhibiting VEGF secretion. In summary, agents are currently available in the clinic that might modulate the TME in a way that could improve radio/chemosensitivity. The challenge is to show that this occurs in human patients and then use this information to optimize cancer therapy.

Treatment and outcomes of acute coronary syndromes in India (CREATE): a prospective analysis of registry data.

BACKGROUND: India has the highest burden of acute coronary syndromes in the world, yet little is known about the treatments and outcomes of these diseases. We aimed to document the characteristics, treatments, and outcomes of patients with acute coronary syndromes who were admitted to hospitals in India. METHODS: We did a prospective registry study in 89 centres from 10 regions and 50 cities in India. Eligible patients had suspected acute myocardial infarction with definite electrocardiograph changes (whether elevated ST [STEMI] or non-STEMI or unstable angina), or had suspected myocardial infarction without ECG changes but with prior evidence of ischaemic heart disease. We recorded a range of clinical outcomes, and all-cause mortality at 30 days. FINDINGS: We enrolled 20,937 patients. Of the 20,468 patients who were given a definite diagnosis, 12,405 (60.6%) had STEMI. The mean age of these patients was 57.5 (SD 12.1) years; patients with STEMI were younger (56.3 [12.1] years) than were those with non-STEMI or unstable angina (59.3 [11.8] years). Most patients were from lower middle 10,737 (52.5%) and poor 3999 (19.6%) social classes. The median time from symptoms to hospital was 360 (IQR 123-1317) min, with 50 (25-68) min from hospital to thrombolysis. 6226 (30.4%) patients had diabetes; 7720 (37.7%) had hypertension; and 8242 (40.2%) were smokers. Treatments for STEMI differed from those for non-STEMI or unstable angina. More patients with STEMI than with non-STEMI were given anti-platelet drugs (98.2%vs 97.4%); angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARB) (60.5%vs 51.2%); and percutaneous coronary interventions (8.0%vs 6.7%, p<0.0001 for all comparisons). Thrombolytics (96.3% streptokinase) were used for 58.5% of patients with STEMI. Conversely, fewer patients with STEMI than those with non-STEMI or unstable angina were given beta blockers (57.5%vs 61.9%); lipid-lowering drugs (50.8%vs 53.9%); and coronary bypass graft surgery (1.9%vs 4.4%, p<0.0001 for all comparisons). The 30-day outcomes for patients with STEMI were death (8.6%), reinfarction (2.3%), and stroke (0.7%). Outcomes for those with non-STEMI or unstable angina were better: death (3.7%), reinfarction (1.2%), and stroke (0.3%, p<0.0001 for all comparisons). Use of key treatments also differed by socioeconomic status: more rich patients than poor patients were given thrombolytics (60.6%vs 52.3%), beta blockers (58.8%vs 49.6%), lipid-lowering drugs (61.2%vs 36.0%), ACE inhibitors or ARB (63.2%vs 54.1%), percutaneous coronary intervention (15.3%vs 2.0%), and coronary artery bypass graft surgery (7.5%vs 0.7%, p<0.0001 for all comparisons). Mortality was higher for poor patients than for rich patients (8.2%vs 5.5%, p<0.0001). Adjustment for treatments (but not risk factors and baseline characteristics) eliminated this difference in mortality. INTERPRETATION: Patients in India who have acute coronary syndromes have a higher rate of STEMI than do patients in developed countries. Since most of these patients were poor, less likely to get evidence-based treatments, and had greater 30-day mortality, reduction of delays in access to hospital and provision of affordable treatments could reduce morbidity and mortality.

Genotypic characterisation of Indian cattle, buffalo and sheep isolates of Echinococcus granulosus.

Twelve isolates of Echinococcus granulosus, collected from domestic animals, including cattle, buffalo and sheep were analysed for DNA nucleotide sequence variation within mitochondrial cytochrome c oxidase I (coxI), NADH dehydrogenase subunit I (nadI) and internal transcribed spacer gene I (ITS1). After analysis of sequence information this was found that the fragment size of ITS1 of buffalo isolate was more in comparison to cattle and sheep isolates. Based on the nadI genotype this was found that Indian cattle, buffalo and sheep isolates could be grouped into E. granulosus sensu stricto. Based on coxI genotype two sheep isolates and one buffalo isolate were homologous to G2 genotype. Rests of the isolates were microvariants of G2 genotype. Presence of G2 genotype in buffalo is the first report of this genotype from this host.

Low pO2 and beta-estradiol induce VEGF in MCF-7 and MCF-7-5C cells: relationship to in vivo hypoxia.

Previous work from this laboratory demonstrated that MCF-7 breast carcinoma cells grown in nude mice contained minimal hypoxia but that tamoxifen treatment of these tumors resulted in increased hypoxia (Evans S. et al., Cancer Research, 1997). These findings led to studies exploring the link between estrogen signaling and tumor oxygenation and determining the role of VEGF in this process. The stimulation of estrogen-dependent MCF-7 breast carcinoma cells in vitro with beta-estradiol resulted in a two-fold induction of VEGF mRNA and 1.3-2-fold increase in protein, similar to what was observed when these cells were exposed to 0. 1% oxygen. Furthermore, the two stimuli given together had an additive effect on (increasing) VEGF expression, suggesting that the combination of hypoxia and estrogen may be important in upregulating VEGF in some breast cancers. Estrogen-independent MCF-7-5C cells, developed by growing MCF-7 cells in long-term culture in estrogen-free media, were also studied. Using EF5, a fluorinated 2-nitroimidazole which localizes to hypoxic cells, MCF-7-5C tumors grown in nude mice were found to contain lower pO2 levels and more hypoxic regions than similarly grown MCF-7 tumors. We tested the hypothesis that this might be the result of defective expression of VEGF in MCF-7-5C cells in response to beta-estradiol and/or hypoxia. However, MCF-7-5C and MCF-7 cells showed a similar induction of VEGF in vitro in response to either beta-estradiol or hypoxia. Therefore, although these two cell lines grown as tumors have substantial differences in the presence and patterns of hypoxia, this could not be explained by a difference in VEGF induction.

Decreased cyclin B1 expression contributes to G2 delay in human brain tumor cells after treatment with camptothecin.

DNA damage produces delayed mitosis (G2/M delay) in proliferating cells, and shortening the delay sensitizes human malignant glioma and medulloblastoma cells to cytotoxic chemotherapy. Although activation of the cyclin-dependent kinase CDC2 mediates G2/M transition in all tumor cells studied to date, regulation of CDC2 varies between tumor types. Persistent hyperphosphorylation of kinase and reduced cyclin expression have been implicated as mediators of treatment-induced G2 delay in different tumor models. To evaluate regulation of G2/M transition in human brain tumors, we studied the expression and/or activity of CDC2 kinase and cyclins A and B1 in U-251 MG and DAOY medulloblastoma cells after their treatment with camptothecin (CPT). Synchronized cells were treated during S phase, then harvested at predetermined intervals for evaluation of cell cycle kinetics, kinase activity mRNA, and protein expression. CPT produced G2 delay associated with decreased CDC2 kinase activity and cyclin B1 expression. Kinase activity was associated with CDC2 bound to cyclin B1, not cyclin A, in both cell lines. Cyclin A mRNA and protein expression were reduced after CPT treatment; however, decreased protein expression was short lived and moderate in the glioma and primitive neuroectodermal tumor/medulloblastoma cells, respectively. We conclude that G2 delay is a common response of brain tumor cells to chemotherapy with topoisomerase I inhibitors and that a mechanism of this delay may be reduced expression of cyclin B1.

Regulation of glut1 mRNA by hypoxia-inducible factor-1. Interaction between H-ras and hypoxia.

Oncogenic transformation and hypoxia both induce glut1 mRNA. We studied the interaction between the ras oncogene and hypoxia in up-regulating glut1 mRNA levels using Rat1 fibroblasts transformed with H-ras (Rat1-ras). Transformation with H-ras led to a substantial increase in glut1 mRNA levels under normoxic conditions and additively increased glut1 mRNA levels in concert with hypoxia. Using a luciferase reporter construct containing 6 kilobase pairs of the glut1 promoter, we showed that this effect was mediated at the transcriptional level. Promoter activity was much higher in Rat1-ras cells than in Rat1 cells and could be down-regulated by cotransfection with a dominant negative Ras construct (RasN17). A 480-base pair (bp) cobalt/hypoxia-responsive fragment of the promoter containing a HIF-1 binding site showed significantly higher activity in Rat1-ras cells than in Rat1 cells, suggesting that Ras might mediate its effect through HIF-1 even under normoxic conditions. Consistent with this, Rat1-ras cells displayed higher levels of HIF1-alpha protein under normoxic conditions. In addition, a promoter construct containing a 4-bp mutation in the HIF1 binding site showed lower activity in Rat1-ras cells than a construct with an intact HIF1 binding site. The activity of the latter construct but not the former could be down-regulated by RasN17, supporting the importance of the HIF1 binding site in regulation by Ras. The phosphatidylinositol 3-kinase inhibitor LY29004 down-regulated glut1 promoter activity and mRNA levels under normoxia and also decreased HIF1alpha protein levels in these cells. Collectively these results indicate that H-Ras up-regulates the glut1 promoter, at least in part, by increasing HIF-1alpha protein levels leading to transactivation of promoter through the HIF-1 binding site.

Hypoxia and VEGF mRNA expression in human tumors.

High expression of circulating plasma vascular endothelial growth factor (VEGF) in patients with cancer is an indicator of poor treatment response. Similarly, hypoxia in tumors, as measured by oxygen needle electrodes, has been found to predict for tumor-treatment failure. These two predictors may be related because hypoxia is a potent stimulator of VEGF expression in vitro. However, the demonstration of a relationship between hypoxia and VEGF in human tumors has, to date, been indirect or even negative. The purpose of this study was to test whether this unexpected result was caused by factors unique to human tumors, or whether the prior results could have been influenced by the known complexities of VEGF regulation. Therefore, we undertook a direct assessment of VEGF induction in human tumors using in situ hybridization and compared its distribution with that of hypoxia, as measured by the distribution of adducts of the hypoxia marker EF5. The distribution of both markers was assessed in relationship to the distribution of blood vessels, as measured by antibodies to CD31. Our hypothesis was that VEGF mRNA and hypoxia would colocalize, assuming that detectability of the former was not limiting. Four squamous cell carcinomas, three sarcomas and one glioblastoma multiforme were studied. When VEGF mRNA signal was detectable, its maxima colocalized with regional maxima of EF5 binding. The strongest levels of both signals were sometimes adjacent to regions of tissue necrosis. However, we were unable to predict absolute levels of EF5 binding based on absolute levels of VEGF mRNA. Conversely, for all tumors studied, regions with relatively low levels of EF5 binding had relatively low or undetectable VEGF mRNA. We found moderate EF5 binding in some keratinized cells but VEGF mRNA was not expressed by these differentiated cells. The paradigm that hypoxia and VEGF expression are linked in human tumors is supported by the data presented herein. A better understanding of the biology behind VEGF expression, including its modulation by hypoxia, is important for optimizing its use as a prognostic indicator and/or modulating its presence with biologic therapies.

Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3'-kinase and distinct from that induced by hypoxia.

Glioblastomas are highly vascular malignant brain tumors that often overexpress vascular endothelial growth factor (VEGF). They also frequently overexpress epidermal growth factor receptor (EGFR) and contain regions of hypoxia, both conditions that can induce VEGF. We examined VEGF regulation in U87 MG human glioblastoma cells and in U87/T691 cells, a clonal derivative that contains a truncated erbB2/Neu receptor that interferes with EGFR signaling through the formation of nonfunctional heterodimeric receptor complexes. U87/T691 cells contained approximately one-half as much VEGF mRNA as did U87 MG cells under normoxic conditions (21% oxygen). Pharmacological inhibition of EGFR, Ras, or PI(3) kinase, but not MAP kinase, led to a significant decrease in VEGF mRNA levels in U87 MG cells. VEGF promoter activity in transient transfections was decreased by either pharmacological or genetic inhibition of EGFR, Ras, or phosphatidylinositol 3'-kinase [PI(3) kinase]. However, inhibition of PI(3) kinase or EGFR did not completely abolish induction of VEGF mRNA by hypoxia (0.2% oxygen). Likewise, VEGF mRNA expression was induced 3-fold by hypoxia in EGFR-inhibited U87/T691 cells, comparable with the fold induction seen in parental U87 MG cells, although the absolute level of message under hypoxia was higher in U87 MG cells. In transient transfections, a luciferase reporter construct containing a 1.2-kb fragment of the VEGF promoter, lacking the known hypoxic-responsive element (HRE), showed up-regulation after EGF stimulation to the same degree as the full-length, 1.5-kb VEGF promoter construct retaining the HRE. Furthermore, activity of the HRE-deleted, 1.2-kb promoter luciferase reporter was down-regulated by PI(3) kinase inhibition. Therefore, in glioblastoma cells, transcriptional regulation of the VEGF promoter by EGFR appears to involve Ras/PI(3) kinase and to be distinct from signals induced by hypoxia.

Both increased stability and transcription contribute to the induction of the urokinase plasminogen activator receptor (uPAR) message by hypoxia.

Both hypoxia and overexpression of the urokinase plasminogen activator receptor (uPAR) are associated with a poor clinical outcome in human cancers. Hypoxia has been shown to induce uPAR expression in breast cancer cells and to increase their invasion through Matrigel, a phenomenon which can be blocked using an anti-uPAR antibody. We examined expression of uPAR mRNA in MCF7 human breast carcinoma cells under hypoxia and found that an increase in the level of the message could be detected at 1% oxygen but was most marked at 0.2 or 0.05% oxygen with an induction of 9- to 20-fold over baseline. To determine whether changes in RNA stability contributed to this dramatic increase, we used actinomycin D to inhibit transcription and found that the half-life of the message was much longer under hypoxic conditions (approximately 10 h) than during reoxygenation (approximately 2 h). Transient transfections using a luciferase reporter construct containing 2 kbp of the mouse uPAR promoter showed that promoter activity increased up to 5-fold after exposure to 0.2% oxygen. Thus, hypoxic induction of the uPAR message in MCF7 cells is due to both mRNA stabilization and increased transcriptional activation.

Identification of putative c-Myc-responsive genes: characterization of rcl, a novel growth-related gene.

The c-Myc protein is a helix-loop-helix leucine zipper oncogenic transcription factor that participates in the regulation of cell proliferation, differentiation, and apoptosis. The biochemical function of c-Myc has been well described, yet the identities of downstream effectors are just beginning to emerge. We describe the identification of a set of c-Myc-responsive genes in the Rat1a fibroblast through the application of cDNA representational difference analysis (RDA) to cDNAs isolated from nonadherent Rat1a and Rat1a-myc cells. In this system, c-Myc overexpression is sufficient to induce the transformed phenotype of anchorage-independent growth. We identified 20 differentially expressed cDNAs, several of which represent novel cDNA sequences. We further characterized one of the novel cDNAs identified in this screen, termed rcl. rcl expression is (i) directly stimulated by c-Myc; (ii) stimulated in the in vivo growth system of regenerating rat liver, as is c-myc; and (iii) elevated in human lymphoid cells that overexpress c-myc. By using an anti-Rcl antibody, immunoblot analysis, and immunofluorescence microscopy, the Rcl protein was found to be a 23-kDa nuclear protein. Ectopic expression of the protein encoded by the rcl cDNA induces anchorage-independent growth in Rat1a fibroblasts, albeit to a diminished extent compared to ectopic c-Myc expression. These data suggest a role for rcl during cellular proliferation and c-Myc-mediated transformation.

Cyclin A message stability varies with the cell cycle.

Progression through the cell cycle in somatic eukaryotic cells is regulated by variations in the levels of cyclin proteins. These protein levels are in turn regulated by cyclical oscillations in their mRNA levels. We show here that regulation of RNA stability plays a role in the mechanisms underlying cell cycle progression. Both cyclin A and B1 messages are expressed at high levels in G2-M and at low levels in early G1. The half-lives of their messages mirror this pattern, long in G2-M (>8 h) and short in early G1 (1-2 h). However, there is evidence of specificity to these changes, because the cyclin A message becomes stable at the G1-S boundary, whereas the cyclin B1 message is unstable until later in S phase. Furthermore, although cyclin B1 mRNA levels are lowered after irradiation because of enhanced instability, cyclin A mRNA levels and message stability are unaffected by irradiation. Additional evidence of specificity was found in an analysis of cyclin E mRNA stability, which remains constant through the cell cycle, although the cyclin E message displays cell cycle-dependent changes in expression. These studies suggest that specific alterations in RNA stability are an important component in regulating the expression of cyclins A and B1 and hence in controlling the cell cycle.

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.

Delayed cyclin B1 expression during the G2 arrest following DNA damage.

Exposure of cells to DNA damaging agents results in a G2 arrest. Exposure of HeLa cells to camptothecin, etoposide or nitrogen mustard for 1 h in S phase resulted in delayed expression of cyclin B1 mRNA during the G2 arrest. Initially the levels of cyclin B1 protein were low as well; however, with extended time the cells blocked in G2 regained higher levels of cyclin B1 protein. In the case of cells treated with nitrogen mustard the higher levels coincided with cells exiting the G2 block into G1. However, with camptothecin or etoposide treatment, while the accumulation of cyclin B1 protein was delayed, its levels eventually surpassed peak levels seen in control cells, in spite of the fact that cells were still blocked in G2. These cells did not continue to progress through the cell cycle indicating further complexity to the mechanisms underlying the G2 block. Decreased transcription and stability of cyclin B1 mRNA were shown to occur after treatment with these DNA damaging agents. These results indicate that suppression of cyclin B1 mRNA expression is one consequence of DNA damage in HeLa cells.

Regulation of radiation-induced apoptosis in oncogene-transfected fibroblasts: influence of H-ras on the G2 delay.

Primary fibroblasts, after serum withdrawal or after irradiation, do not undergo apoptosis. Myc-transfected fibroblasts, in contrast, undergo apoptosis upon serum withdrawal and after irradiation. We have studied the relationship of apoptosis induction to effects on the G2 phase cell cycle in a series of rat embryo cells transformed by rasH plus myc or immortalized by myc alone. In this system, while the presence of rasH had little effect on the extent of apoptosis induction by serum withdrawal, rasH greatly suppressed the apoptotic response of myc-transfected cells to X-rays. The cells into which rasH had been introduced showed a profound G2 arrest associated with suppression of cyclin B1 mRNA expression. In contrast, cells with myc alone had a minimal G2 delay after irradiation and no suppression of cyclin B1 mRNA expression. We hypothesize that rasH, by influencing the G2 response of cells to X-rays, exerts an anti-apoptotic effect. In support of this hypothesis; we found that treatment of cells with caffeine, an agent that relieves the G2 delay after irradiation resulted in increased apoptosis in the irradiated cells, but not in control cells.