Harris Bernstein

DNA repair/pro-apoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis

Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication past DNA damages, causing mutations leading to carcinogenesis. It has recently become apparent that key proteins which contribute to cellular survival by acting in DNA repair become executioners in the face of excess DNA damage. Five major DNA repair pathways are homologous recombinational repair (HRR), non-homologous end joining (NHEJ), nucleotide excision repair (NER), base excision repair (BER) and mismatch repair (MMR). In each of these DNA repair pathways, key proteins occur with dual functions in DNA damage sensing/repair and apoptosis. Proteins with these dual roles occur in: (1) HRR (BRCA1, ATM, ATR, WRN, BLM, Tip60 and p53); (2) NHEJ (the catalytic subunit of DNA-PK); (3) NER (XPB, XPD, p53 and p33(ING1b)); (4) BER (Ref-1/Ape, poly(ADP-ribose) polymerase-1 (PARP-1) and p53); (5) MMR (MSH2, MSH6, MLH1 and PMS2). For a number of these dual-role proteins, germ line mutations causing them to be defective also predispose individuals to cancer. Such proteins include BRCA1, ATM, WRN, BLM, p53, XPB, XPD, MSH2, MSH6, MLH1 and PMS2.

DNA Damage as the Primary Cause of Aging

DNA damage appears to be ubiquitous in the biological world, as judged by the variety of organisms which have evolved DNA-repair systems. Previously, it was proposed that germ-line DNA of multicellular organisms may be protected from damage, and consequently from aging, by efficient recombinational repair during meiosis. The somatic line, however, may be vulnerable to the accumulation of DNA damage, and hence undergo aging, owing to relatively less repair. Although DNA Lesions most important in aging are not known yet, these is evidence for several types of endogenous damage. DNA lesions have been shown to interfere with transcription and replication, and so lead to loss of cell function and death. In mammals, there is a progressive decline of function in many different tissues with increasing age. Deterioration of central nervous system functions appears to be a critical part of the aging process. This may be due to the low DNA repair capacity which is found in postmitotic brain tissue, and which could result in the accumulation of DNA lesions in this tissue. Also reviewed is evidence that species longevity is directly related to tissue DNA-repair capacity and that aging may be accelerated by treatment with DNA-damaging agents, or in individuals with genetically defective repair. Although it has been frequently postulated that somatic mutation may be a cause of aging, current evidence suggests that it is probably less important than DNA damage. A prominent theory on the evolution of aging, which attributes special importance to genes that are advantagous in youth but are deleterious later on, is discussed in terms of regulatory genes that reduce DNA repair as cells differentiate to the postmitotic state. Finally, we hypothesize that the factors which determine maximum longevity of individuals in a population are the rate of occurrence of DNA damage, the rate of DNA repair, the degree of cellular redundancy, and the extent of exposure to stress.

Apoptosis Overview Emphasizing the Role of Oxidative Stress, DNA Damage and Signal- Transduction Pathways

Apoptosis (programmed cell death) is a central protective response to excess oxidative damage (especially DNA damage), and is also essential to embryogenesis, morphogenesis and normal immune function. An understanding of the cellular events leading to apoptosis is important for the design of new chemotherapeutic agents directed against the types of leukemias and lymphomas that are resistant to currently used chemotherapeutic protocols. We present here a review of the characteristic features of apoptosis, the cell types and situations in which it occurs, the types of oxidative stress that induce apoptosis, the signal-transduction pathways that either induce or prevent apoptosis, the biologic significance of apoptosis, the role of apoptosis in cancer, and an evaluation of the methodologies used to identify apoptotic cells. Two accompanying articles, demonstrating classic apoptosis and non-classic apoptosis in the same Epstein-Barr virus-transformed lymphoid cell line, are used to illustrate the value of employing multiple criteria to determine the type of cell death occurring in a given experimental system. Aspects of apoptosis and programmed cell death that are not covered in this review include histochemistry, details of cell deletion processes in the sculpting of tissues and organs in embryogenesis and morphogenesis, and the specific pathways leading to apoptosis in specific cell types. The readers should refer to the excellent books and reviews on the morphology, biochemistry and molecular biology of apoptosis already published on these topics. Emphasis is placed, in this review, on a proposed common pathway of apoptosis that may be relevant to all cell types.

Nicotine increases oxidative stress, activates NF-κB and GRP78, induces apoptosis and sensitizes cells to genotoxic/xenobiotic stresses by a multiple stress inducer, deoxycholate: relevance to colon carcinogenesis

Epidemiologic studies indicate that environmental (smoking) and dietary factors (high fat) contribute to carcinogenesis in many organ systems. The aim of our study was to test the hypothesis that nicotine, a component of cigarette smoke, and sodium deoxycholate (NaDOC), a cytotoxic bile salt that increases in concentration in the gastrointestinal tract after a high fat meal, induce similar cellular stresses and that nicotine may enhance some of the NaDOC-induced stresses. We found that nicotine, at 0.8 microM, the very low sub-micromolar level occurring in the tissues of smokers: (1). increases oxidative stress; (2). activates NF-kappaB, a redox-sensitive transcription factor; (3). activates the 78 kD glucose regulated protein promoter, an indication of endoplasmic reticulum stress; (4). induces apoptosis; (5). enhances the ability of NaDOC to activate the 153 kD growth arrest and DNA damage promoter, an indication of increased genotoxic stress; and (6). enhances the ability of NaDOC to activate the xenobiotic response element. Our findings have applicability to G.I. cancer, in general, since smoking is a risk factor in the development of esophageal, pancreatic, gastric and colon cancer, and these cancers are also promoted by bile acids.

Oxidative and other DNA damages as the basis of aging: a review.

DNA damages occur continuously in cells of living organisms. While most of these damages are repaired, some accumulate. In particular, there is evidence for DNA damage accumulation in non-dividing cells of mammals. These accumulated DNA damages probably interfere with RNA transcription. We consider that the decline in the ability of DNA to serve as a template for gene expression is the primary cause of aging. Oxidative DNA damages are among the best documented and prevalent DNA damages and are likely to be a prominent cause of aging.

Activation of the promoters of genes associated with DNA damage, oxidative stress, ER stress and protein malfolding by the bile salt, deoxycholate

Toxic bile salts, retained within the liver because of impaired biliary excretion, are considered to play a major role in liver injury during cholestasis. Bile salts cause cellular stresses that may result in apoptosis. To better understand such cellular stresses, the effect of the bile salt sodium deoxycholate (NaDOC) on activation of 13 specific gene promoters or response elements associated with different cellular stresses was measured in the transformed human hepatoma line, HepG2. NaDOC was found to activate transcription factors and induce or activate the promoters of genes that respond to protein malfolding (grp78 and hsp70), DNA damage (gadd153, hsp70 and c-fos), oxidative stress (NF-kappaB, c-fos, hsp70 and gadd153), ER stress (grp78) and Ca++ imbalance (grp78).

The stress-response proteins poly(ADP-ribose) polymerase and NF-κB protect against bile salt-induced apoptosis

Bile salts induce apoptosis and are implicated as promoters of colon cancer. The mechanisms by which bile salts produce these effects are poorly understood. We report that the cytotoxic bile salt, sodium deoxycholate (NaDOC), activates the key stress response proteins, NF-κB and poly(ADP-ribose) polymerase (PARP). The activation of NF-κB and PARP, respectively, indicates that bile salts induce oxidative stress and DNA damage. The pre-treatment of cells with specific inhibitors of these proteins [pyrrolidine dithiocarbamate (NF-κB inhibitor) and 3-aminobenzamide (PARP inhibitor)] sensitizes cells to the induction of apoptosis by NaDOC, indicating that these stress response pathways are protective in nature. Colon cancer risk has been reported to be associated with resistance to apoptosis. We found an increase in activated NF-κB at the base of human colon crypts that exhibit apoptosis resistance. This provides a link between an increased stress response and colon cancer risk. The implications of these findings with respect to apoptosis and to colon carcinogenesis are discussed.

EVOLUTION OF SEXUAL REPRODUCTION: IMPORTANCE OF DNA REPAIR, COMPLEMENTATION, AND VARIATION

We have proposed that sexual reproduction arose in evolution as a DNA repair process which allowed damage in one chromosome to be repaired by the information in another homologous chromosome, and has retained this advantage throughout evolution. Since this process required that two chromosomes be present in a common cytoplasm, additional ways were evolved to take advantage of the redundant information available. The diploid stage of the cycle was probably transient in early organisms, but began to take on a more significant role as genome size increased, since it provided protection against the expression of deleterious mutations. As the diploid stage of the sexual cycle became the predominant stage, genome information content expanded beyond the range of haploid organisms. The shift to diploidy is essentially irreversible, and likewise helps prevent the abandonment of sexual reproduction. A consequence of the DNA exchange reactions that constitute recombinational repair is the reassortment of parental genetic information among progeny. As is widely appreciated, this variation is advantageous in itself under certain conditions. However we argue that a complete evolutionary explanation for the maintenance of sexual reproduction should also include complementation and DNA repair.

Role of Apoptosis in Biology and Pathology: Resistance to Apoptosis in Colon Carcinogenesis

The overview of apoptosis presented here emphasizes cell deletion in the immune system, with particular reference to T- and B-lymphocyte development, and the in vivo and in vitro senescence of human neutrophils. Some biochemical criteria that are used to identify apoptotic cells are described. Pitfalls in using agarose gel electrophoresis as the sole method for the identification of apoptotic cells are discussed. There are multiple modes of cell death that can be identified at the morphologic level. Thus the central role of microscopic methods, and in particular, electron microscopy, as an important tool in the study of cell death mechanisms, is presented. Apoptosis has a protective role against disease and could, a priori, have an important role in either the initiation or progression of cancer. Two paradoxes concerning the relationship of tumor aggressiveness at the clinical level to mitotic activity have been explained by an evaluation of apoptotic index. In the first case, basal cell carcinomas grow slowly but show a high rate of mitosis. Here, the apoptotic rate is quite high, but just below the mitotic rate, thereby accounting for the slow rate of growth. A second instance is follicular lymphoma, which has a low rate of mitosis that is less than that described for reactive germinal centers. However, apoptosis is markedly reduced in follicular lymphomas compared with that seen in reactive germinal centers, thus providing an explanation for the progressive growth of the follicle. We present a brief description of recent work from our laboratory that indicates that apoptosis may play an important role in colon carcinogenesis. We have shown that sodium deoxycholate, the particular bile salt present in highest concentration in the colon, induces apoptosis in the goblet cells of the human colonic mucosa in an in vitro assay. The intriguing finding is that cells of the normal-appearing mucosa of colon cancer patients are resistant to bile salt-induced apoptosis. This suggests a novel hypothesis about the etiologic role of bile salts in colon cancer. The chronic presence of bile salts that accompany a high-fat diet could select for apoptosis-resistant epithelial cells in the colon over time. Thus, a resistance-to-apoptosis bioassay may prove useful as an intermediate biomarker for determining which individuals are at high risk for colon cancer.

The NAD + precursors, nicotinic acid and nicotinamide protect cells against apoptosis induced by a multiple stress inducer, deoxycholate

The bile salt, sodium deoxycholate (NaDOC), is a natural detergent that promotes digestion of fats. At high physiologic levels, NaDOC activates many stress-response pathways and induces apoptosis in various cell types. NaDOC induces DNA damage and activates poly(ADP-ribose) polymerase (PARP), an enzyme that utilizes NAD+ as a substrate to repair DNA. NaDOC also induces oxidative stress, endoplasmic reticulum (ER) stress and contributes to protein malfolding. The NAD+ precursors, nicotinic acid (NA) and nicotinamide (NAM) were found to protect cells against NaDOC-induced apoptosis. NA and NAM also decreased constitutive levels of both activated NF-κB and GRP78, two proteins that respond to oxidative stress. However, the mechanism by which NA and NAM protects cells against apoptosis does not involve a reduction in constitutive levels of oxidative stress. NA or NAM treatment increased the protein levels of glyceraldehyde-3-phosphate dehydrogense (GAPDH), a multi-functional enzyme, in the nucleus and cytoplasm, respectively. NAM did not activate the promoter/response elements of 13 stress response genes nor reduce intracellular non-protein thiols, suggesting that it is non-toxic to cells. NAM thus has promise as a dietary supplement to help prevent disorders involving excessive apoptosis.