Cell cycle regulation of the human DNA mismatch repair genes hMSH2, hMLH1, and hPMS2.

Hereditary nonpolyposis colorectal cancer is a cancer susceptibility syndrome that has been found to be caused by mutations in any of several genes involved in DNA mismatch repair, including hMSH2, hMLH1, or hPMS2. Recent reports have suggested that hMSH2 and hMLH1 have a role in the regulation of the cell cycle. To determine if these genes are cell cycle regulated, we examined their mRNA and protein levels throughout the cell cycle in IMR-90 normal human lung fibroblasts. We demonstrate that the levels of hMSH2 mRNA and protein do not change appreciably throughout the cell cycle. Although hMLH1 mRNA levels remained constant, there was a modest (approximately 50%) increase in its protein levels during late G1 and S phase. The levels of hPMS2 mRNA fluctuated (decreasing 50% in G1 and increasing 50% in S phase), whereas hPMS2 protein levels increased 50% in late G1 and S phase. Our data indicate that, at least in normal cells, the machinery responsible for the detection and repair of mismatched DNA bases is present throughout the cell cycle.

DNA-dependent protein kinase does not play a role in adaptive survival responses to ionizing radiation.

We previously demonstrated that exposure of certain human tumor cells to very low chronic doses of ionizing radiation led to their enhanced survival following exposure to subsequent high doses of radiation. Survival enhancement due to these adaptive survival responses (ASRs) ranged from 1.5-fold to 2.2-fold in many human tumor cells. Furthermore, we showed that ASRs result from altered G1 checkpoint regulation, possibly mediated by overexpression of cyclin D1, proliferating cell nuclear antigen (PCNA), and the X-ray induction of cyclin A. Because cyclin D1 and PCNA proteins are components of many DNA synthetic and repair processes in the cell, we tested the hypothesis that preexposure of cells to low doses of ionizing radiation enabled activation of the DNA repair machinery needed for survival recovery after high-dose radiation. We examined the role of DNA break repair in ASRs using murine cells deficient (i.e., severe combined immunodeficiency [SCID] cells) or proficient (i.e., parental mouse strain [CB-17] cells) in DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression and DNA double-strand break repair, DNA-PKcs is a nuclear serine/threonine protein kinase that is activated by DNA breaks and plays a key role in double-strand break repair. DNA-PKcs also phosphorylates several nuclear DNA-binding regulatory transcription factor proteins (e.g., Sp1 and p53), which suggests that DNA-PKcs may play a role in regulating transcription, replication, and recombination as well as DNA repair, after radiation. Therefore, we exposed confluent SCID or CB-17 cells to low priming doses of ionizing radiation (i.e., 5 cGy) and compared the survival responses of primed cells to those of unprimed cells after an equitoxic high-dose challenge. Low-dose-primed SCID or CB-17 cells demonstrated 2-fold enhanced survival after a high-dose challenge compared to that of unprimed control cells. These data suggest that expression of the catalytic subunit of DNA-PKcs (expressed in CB-17 not SCID cells) and the presence of active double-strand break repair processes (active in CB-17, deficient in SCID cells) do not play a major role in ASRs in mammalian cells. Furthermore, we present data that suggest that DNA-PKcs plays a role in the regulation of the G2/M cell cycle checkpoint following extremely high doses of ionizing radiation.

The behaviour of normal and agravitropic transgenic roots of rapeseed (Brassica napus L.) under microgravity conditions.

In the TRANSFORM experiment for IML-2 on the Space Shuttle Columbia, normal (wild type = WT) and genetically transformed agravitropic rapeseed roots were tested under microgravity conditions. The aim of the experiment was to determine if the wild-type roots behaved differently (growth, morphology, gravitropical sensitivity) from the transgenic roots. The appearance of the organelles and distribution of statoliths (i.e. amyloplasts with starch grains) in the gravitropic reactive cells (statocytes) under weightlessness was compared for the two types of roots. Attempts have also been made to regenerate new plants from the root material tested in space. Both the WT and the transgenic root types showed the expected increase in length during 36 h of photorecording. Contrary to the results of the ground controls, no significant difference in elongation rates was found between the WT and transgenic roots grown in orbit. However, there are indications that the total growth both in the WT and the transgenic roots was higher in the ground control than for roots in orbit. After a 60 min 1 x g stimulation of the roots on board the Shuttle, no detectable curvatures were obtained in either the transgenic or the WT roots. However, it cannot be excluded that a minute curvature development occurs in the root tips but was not detected due to technical reasons. The ultrastructure was well preserved in both the WT and the transgenic roots, despite the fact that the tissue was kept in the prefixative for over 3 weeks. No marked differences in ultrastructure were observed between the transformed root statocyte cells and the equivalent cells in the wild type. There were no obvious differences in root morphology during the orbital period. Light micrographs and morphometrical analysis indicate that the amyloplasts of both the wild type and transformed root statocytes are randomly distributed over the cells kept under micro-g conditions for 37 h after a 14 h stimulation on the 1 x g centrifuge. The main scientific conclusion from the TRANSFORM experiment is that the difference in growth found in the ground control between the WT and the transgenic root types seems to be eliminated under weightlessness. Explanations for this behaviour cannot be found in the root ultrastructure or in root morphology.

Altered G1 checkpoint control determines adaptive survival responses to ionizing radiation.

Adaptive survival responses (ASRs) are observed when cells become more resistant to a high dose of a cytotoxic agent after repeated low dose exposures to that agent or another genotoxic agent. Confluent (G0/G1) human normal (GM2936B, GM2937A, AG2603, IMR-90), cancer-prone (XPV2359), and neoplastic (U1-Mel, HEp-2, HTB-152) cells were primed with repeated low doses of X-rays (ranging from 0.05-10 cGy/day for 4 days), then challenged with a high dose (290-450 cGy) on day 5. U1-Mel and HEp-2 cells showed greater than 2-fold transient survival enhancement when primed with 1-10 cGy. ASRs in U1-Mel or HEp-2 cells were blocked by cycloheximide or actinomycin D. Increases in cyclins A and D1 mRNAs were noted in primed compared to unirradiated U1-Mel and HEp-2 cells; however, only cyclin A protein levels increased. Cyclin D1 and proliferating cell nuclear antigen (PCNA) protein levels were constitutively elevated in HEp-2 and U1-Mel cells, compared to the other human normal and neoplastic cells examined, and were not altered by low or high doses of radiation. Low dose primed U1-Mel cells entered S-phase 4-6 h faster than unprimed U1-Mel cells upon low-density replating. Similar responses in terms of survival recovery, transcript and protein induction, and altered cell cycle regulation were not observed in the other human normal, cancer-prone or neoplastic cells examined. We hypothesize that only certain human cells can adapt to ionizing radiation by progressing to a point later in G1 (the A point) where DNA repair processes and radioresistance can be induced. ASRs in human cells correlated well with constitutively elevated levels of PCNA and cyclin D1, as well as inducibility of cyclin A. We propose that a protein complex composed of cyclin D1, PCNA, and possibly cyclin A may play a role in cell cycle regulation and DNA repair, which determine ASRs in human cells.

Molecular analyses of adaptive survival responses (ASRs): role of ASRs in radiotherapy.

Adaptive survival responses (ASRs), whereby cells demonstrate a survival advantage when exposed to very low doses of ionizing radiation (IR) 4 - 24 h prior to a high dose challenge, were first reported over 15 years ago. These responses were linked to hormesis, which implied that exposure to low levels of IR may be beneficial to the cell. We postulate that increased survival does not necessarily mean that the treatment is beneficial. Studies at the molecular level indicate that ASRs are the result of misregulated cell cycle checkpoint responses, occurring in the G1 phase of the cell cycle after IR. Specific gene products (i.e., PCNA, cyclin D1, cyclin A, XIP8, xip5 and xip13) appear to control these cell cycle checkpoint responses. Certain neoplastic cells show potent ASRs because they bypass checkpoints which would otherwise lead to apoptosis or other forms of cell death (possibly necrosis), and/or these cancer cells lack genetic factors, such as specific caspases (cysteine aspartate-specific proteases), that control apoptosis. Alterations in these cell cycle checkpoints or apoptotic responses may also occur during IR-induced stress responses in normal cells, at critical times (10-18 days posttreatment) following IR. One IR-induced protein, XIP8, may be a critical controlling factor at this point where delayed-onset apoptosis occurs. Additionally, we have shown that the presence or absence (i.e., SCID cells) of nonhomologous DNA double strand break repair did not seem to influence ASRs, suggesting that ASRs may be caused by signal transduction stress responses. ASRs may be beneficial to survival, however, the consequence(s) of that survival may be dire. For example, many neoplastic cells exhibited far greater ASRs than normal cells. Additionally, ASRs were induced by as little as 1 cGy and and were enhanced by repeated exposures of low level radiation. The implications for radiotherapy are that when a patient arrives for port film imaging during the course of therapy, the dose-rate, overall level of exposure, and time between port film exposure and high dose IR treatment become potentially important factors for improved efficacy of treatment of certain cancers. Further research is warranted to determine what molecular factors are most important for ASRs, and current work is focusing on XIP8.

Cardiac tamponade as a result of infusion therapy. A potentially amenable complication of central venous catheters.

A fatal case of infusion of a fat emulsion (Intralipid) into the pericardium is reported. Perforation of the anterior wall of the right ventricle of the heart by a central venous catheter had occurred 3 days after insertion via the basilic vein. Local myocardial inflammation and necrosis along the puncture wound through the myocardium was a feature. The incident underlines the necessity for prompt assurance of correct positioning of central venous catheters. It is emphasized that effective treatment is possible if one is aware of some important features of the condition. Immediate diagnosis is mandatory.

[Reports of waiting list data from hospitals to the central registry--a database for reducing hospital waiting lists. Minimum dataset of waiting list parameters]. / Rapportering av ventelistedata fra sykehus til sentralregisteret--database for reduksjon av sykehuskø. Minste felles datasett med ventelisteparametre.

The governmental regulations concerning registration of waiting lists and priority of patients, laid down in July 1990, introduce a "waiting time guarantee" which ensures a waiting time not exceeding six months for patients suffering from diseases having severe impacts on health. Hospitals that are unable to treat these patients within six months are requested to refer them to other hospitals before the deadline. All hospitals have to make monthly reports of waiting list parameters to a Central Waiting List Register, enabling both a nationwide waiting list survey and comparisons between different hospitals and different counties. An online communication facility to the central register enables searches for and reporting of vacant treatment capacity.

Agravitropic behaviour of roots of rapeseed (Brassica napus L.) transformed by Agrobacterium rhizogenes.

Transgenic hairy roots of Brassica napus (cv. Omega) have been developed, using Agrobacterium rhizogenes strain AR 25, for use as a model system in the investigation of physiological and morphological differences between transgenic and normal roots. The basic parameters of growth and normal or altered gravitropical behaviour of hairy roots are for the first time presented in this paper together with an ultrastructural and morphological analysis of the root statocytes. The results obtained also represented the basis for the TRANSF0RM-experiment on the IML-2 mission performed onboard the Space Shuttle Columbia. Typical hairy root traits such as hormone-autonomous growth high growth rate, lateral branching, and changed/absence of gravitropism were detected. The transformed nature of the roots was confirmed by Southern blot analyses. The gravitropical behaviour of apices from hairy root cultures of this clone has been compared with root tips from normal seedlings. While the wild type roots curved progressively with increasing stimulation angles, the transformed roots showed no curvature when stimulated at 45 degrees, 90 degrees or 135 degrees on the ground. The morphology and ultrastructure of the root tip regions were examined by light microscopy and transmission electron microscopy. At the ultrastructural level no major differences could be detected between the roots studied. There was, however, a slight reduction in the starch content of most of the amyloplasts of the transgenic root tips, and the root cap was more V-shaped in the transgenic roots than in the wild type. Preliminary results from the Shuttle experiment TRANSFORM show a random distribution of amyloplasts in the root cells of both transformed and wild type root caps after 14 h on a 1xg centrifuge followed by 37 h in microgravity.

Isolation of Ku70-binding proteins (KUBs).

DNA-dependent protein kinase (DNA-PK) plays a critical role in resealing DNA double-stand breaks by non-homologous end joining. Aside from DNA-PK, XRCC4 and DNA ligase IV, other proteins which play a role(s) in this repair pathway remain unknown; DNA-PK contains a catalytic subunit (DNA-PKcs) and a DNA binding subunit (Ku70 and Ku80). We isolated Ku70-binding proteins (KUB1-KUB4) using yeast two-hybrid analyses. Sequence analyses revealed KUB1 to be apolipoprotein J (apoJ), also known as X-ray-inducible transcript 8 (XIP8), testosterone-repressed prostate message-2 (TRPM-2) and clusterin. KUB2 is Ku80. KUB3 and KUB4 are unknown, >10 kb trans-cripts. Interactions of apoJ/XIP8 or KUB3 with Ku70 were confirmed by co-immunoprecipitation analyses in MCF-7:WS8 breast cancer or IMR-90 normal lung fibroblast cells, respectively. The interaction of apoJ/XIP8 with Ku70 was confirmed by far-western analyses. Stable over-expression of full-length apoJ/XIP8 in MCF-7:WS8 caused decreased Ku70/Ku80 DNA end binding that was restored by apoJ/XIP8 monoclonal antibodies. The role of apoJ/XIP8 in ionizing radiation resistance/sensitivity is under investigation.

Damage-sensing mechanisms in human cells after ionizing radiation.

Human cells have evolved several mechanisms for responding to damage created by ionizing radiation. Some of these responses involve the activation or suppression of the transcriptional machinery. Other responses involve the downregulation of enzymes, such as topoisomerase I, which appear to be necessary for DNA repair or apoptosis. Over the past five years, many studies have established links between DNA damage, activation of transcription factors that are coupled to DNA repair mechanisms, increased gene transcription and altered cell cycle regulation to allow for repair or cell death via apoptosis or necrosis. Together these factors determine whether a cell will survive with or without carcinogenic consequences. The immediate responses of human cells to ionizing radiation, in terms of sensing and responding to damage, are therefore, critical determinants of cell survival and carcinogenesis.