Differential regulation of calcitonin secretion in normal and neoplastic pulmonary neuroendocrine cells in vitro.

Within the mammalian lung, cells with a neuroendocrine phenotype are few in number and are sparsely distributed. In contrast, neuroendocrine neoplasms represent a major group of lung cancers. The aim of this study was to develop a model of mammalian PNECs and to compare glucocorticoid regulation of calcitonin secretion in normal and neoplastic cells with neuroendocrine differentiation. Cell cultures of PNECs were initiated after the disaggregation of neonatal hamster lungs with 0.1% collagenase and fractionation of the resultant cell suspension on a gradient of iodixanol (1.320 g/mL). Cell fractions enriched in PNECs were identified by positive staining for 5-hydroxytryptamine and the presence of calcitonin. Calcitonin secretion was investigated after exposure to hydrocortisone (0 to 1,000 nM). A dose-dependant inhibition of calcitonin secretion was seen after 7 days between 10 nM (55% of control), and 1,000 nM (29%) hydrocortisone. Cell cultures grown in the presence of hydrocortisone also contained significantly fewer PNECs between 10 nM (90% of control), and 1,000 nM (45%). Human bronchial carcinoid cells (NCIH727) cultured under identical conditions showed a similar inhibition of calcitonin secretion between 10 nM (53%) and 1,000 nM (52%), although at these concentrations, no reduction in cell number was seen. In contrast, 2 human small cell lung cancer cell lines (DMS-79 and COR-L24 cells) showed no dose-dependent inhibition of calcitonin secretion and no effect on cell proliferation in response to hydrocortisone. These results show that enriched cultures of mammalian PNECs can be used to investigate functional aspects of their biology, including peptide secretion in response to potential regulators. Furthermore, calcitonin secretion is inhibited in normal PNECs and bronchial carcinoid cells at physiological concentrations of glucocorticoids, but this feature appears not to be present in the 2 more invasive neuroendocrine neoplasms (small cell lung cancer cells) investigated in this study.

DNA repair: kinetics and thresholds.

DNA damage is a critical factor in the initiation of chemically induced toxicities (including cancer), and the repair of this damage represents the cell's first line of defense against the deleterious effects of these agents. The various mechanisms of DNA repair are reviewed briefly and the actions of the DNA repair protein O6-alkylguanine DNA alkyltransferase (ATase) are used to illustrate how DNA repair can protect cells against alkylating agent-induced toxicities, mutagenesis, clastogenesis, and carcinogenesis. The effectiveness of this repair protein can be measured based on its ability to deplete levels of its promutagenic substrate O6-methylguanine (O6-meG) in the DNA of cells. These studies reveal that the repair of O6-meG from DNA occurs heterogeneously, both intra- and intercellularly. Even in cells that repair O6-meG hyperefficiently, certain regions of chromatin DNA are repaired with difficulty, and in other regions they are not repaired at all; most likely this lack of repair is a result of the location of the lesion in the DNA sequence. When individual cells are compared within a tissue, some cells are clearly repair deficient, because the O6-meG can persist in DNA for many weeks, whereas in other cells, it is removed within a matter of hours. The role of these repair-deficient cells as targets for alkylating agent induced carcinogenesis is considered. The mechanisms of the homeostatic control of DNA repair function in mammalian cells are not yet well understood. Because there are now indications of the mechanisms by which the level of DNA damage may be sensed (and so influence the activity of the ATase repair protein), this is an important area for future study.

Overexpression of the glucose-regulated stress gene GRP78 in malignant but not benign human breast lesions.

The 78 kDa glucose-regulated stress protein GRP78 is induced by physiological stress conditions such as hypoxia, low pH, and glucose deprivation which often exist in the microenvironments of solid tumors. Activation of this stress pathway occurs in response to several pro-apoptotic stimuli. In vitro studies have demonstrated a correlation between induced expression of GRP78 and resistance to apoptotic death induced by topoisomerase II-directed drugs. We were interested in characterizing this protein in human breast lesions for potential implications in chemotherapeutic intervention. Surgical specimens of human breast lesions and paired normal tissues from the same patients were flash frozen for these studies. Total RNA and/or protein were extracted from these tissues and used in northern and/or western blot analyses, respectively, to quantify the relative expression of GRP78. Northern blot analysis indicated that 0/5 benign breast lesions, 3/5 estrogen receptor positive (ER+) breast tumors, and 6/9 estrogen receptor negative (ER-) breast tumors exhibited overexpression of GRP78 mRNA compared to paired normal tissues, with fold overexpressions ranging from 1.8 to 20. Western blot analyses correlated with these findings since 0/5 benign breast lesions, 4/6 ER+ breast tumors, and 3/3 ER- breast tumors overexpressed GRP78 protein with fold overexpressions ranging from 1.8 to 19. Immunohistochemical analysis of these tissues demonstrated that the expression of GRP78 was heterogeneous among the cells comprising different normal and malignant glands, but confirmed the overexpression of GRP78 in most of the more aggressive ER- tumors. These results suggest that some breast tumors exhibit adverse microenvironment conditions that induce the overexpression of specific stress genes that may play a role in resistance to apoptosis and decreased chemotherapeutic efficacy.

Arrest of replication by mammalian DNA polymerases alpha and beta caused by chromium-DNA lesions.

We have previously shown that trivalent chromium, and hexavalent chromium in the presence of one of its primary in vivo reductants, ascorbate, can bind to DNA and form interstrand crosslinks capable of obstructing replication. This effect was demonstrated in vitro by using Sequenase Version 2.0 T7 DNA polymerase; its parent enzyme, the unmodified T7 DNA polymerase; and Escherichia coli polymerase I large (Klenow) fragment; and it was demonstrated ex vivo by using Taq polymerase and DNA from chromium-treated human lung cells as template. This study was performed to determine whether DNA-bound chromium affects mammalian DNA polymerases in the same manner. Two mammalian enzymes, DNA polymerase alpha and DNA polymerase beta, were used. DNA polymerase alpha is a processive enzyme believed to be the primary lagging-stand synthetase, whereas DNA polymerase beta is a non-processive enzyme believed to function in DNA repair by filling single stranded gaps one base at a time. DNA polymerase arrest assays were performed with each of these enzymes to replicate DNA with toxicologically relevant levels of chromium adducts produced by either trivalent chromium or hexavalent chromium and ascorbate. Both enzymes responded to chromium-DNA damage by arresting replication, and the arrests increased in a dose-dependent manner. Furthermore, the guanine-specific pattern of arrests produced when an exonuclease-free preparation of DNA polymerase beta was used corresponded exactly to the arrest patterns produced in vitro by the exonuclease-free enzyme Sequenase and ex vivo by Taq polymerase. These results suggest that replication arrest may be a common response of polymerases to DNA-chromium lesions and provide a plausible mechanism for the inhibition of DNA synthesis and S-phase cell-cycle delay that occurs in mammalian cells treated with genotoxic chromium compounds.

Apoptosis: inhibitor or instigator of carcinogenesis?

Carcinogenesis is considered to require an initiating event that results in an irreversible genetic change in a subpopulation of cells. Based on the available evidence, it seems likely that apoptosis may act to attenuate this process by causing the deletion of genetically damaged cells from the host organism. Nevertheless, the existence of an active pathway leading to apoptotic cell death may be a double-edged sword, simply because it can be overcome. Some cells may exhibit preexisting genetic or epigenetic insensitivity to induction of apoptosis. Surviving cells may contain sub- lethal levels of DNA damage and be induced to proliferate as an indirect result of the carcinogen-induced apoptotic cell death of surrounding tissue. This process would facilitate the acquisition mutations in the genome, possibly resulting in further insensitivity to apoptosis through activation of the bcl-2 oncogene or inactivation of the p53 tumor suppressor gene. In this context, the propensity of a cell to undergo apoptosis could be viewed as a selection pressure that a tumor cell must overcome. For neoplastic growth to occur, an imbalance between proliferation and apoptosis must be established such that cell growth predominates. Genetic mutations or epigenetic factors that diminish the propensity of a cell to undergo apoptosis may therefore confer on that cell a growth advantage.

Base-specific arrest of in vitro DNA replication by carcinogenic chromium: relationship to DNA interstrand crosslinking.

We have previously shown that trivalent chromium can bind to purified DNA and form lesions capable of obstructing DNA replication in vitro. Trivalent chromium is not, however, carcinogenic to humans. Rather, it is the end product of the intracellular reduction of hexavalent chromium, which is carcinogenic. The process of chromium reduction yields several reactive intermediates which may also interact with DNA, perhaps producing different lesions than those generated when trivalent chromium binds DNA. The present study was undertaken to determine whether the treatment of DNA with hexavalent chromium in the presence of ascorbate (the intracellular reductant responsible for most in vivo chromium reduction), would also generate DNA lesions capable of obstructing replication. Using increasing chromium concentrations and a constant ascorbate:chromium ratio of 0.5:1 to generate biologically relevant adduct levels, a DNA polymerase arrest assay revealed that polymerase arresting lesions were formed and were indistinguishable from those generated by trivalent chromium, in that the most prominent arrests sites were one base upstream of guanine residues on the template strand. Measurement of the amount of chromium bound to template DNA in relation to the number of arrests demonstrated that only a subset (18.5%) of the chromium adducts were capable of causing polymerase arrest. Arrest assays performed with increasing ratios of ascorbate to chromium showed that high ratios (> or = 5:1) resulted in decreased polymerase arrests. DNA interstrand crosslinks in the arrest assay template were detected by renaturing agarose gel electrophoresis, and were shown to decrease markedly with increasing ascorbate to chromium ratios, whereas chromium binding levels remained unchanged. These results strongly implicate DNA interstrand crosslinks as the polymerase arresting lesion. The present study confirms and extends our previous study with trivalent chromium, and suggests that while the initial chemical nature of the DNA lesions formed by either trivalent chromium or reductive intermediates of hexavalent chromium may differ, their effect on DNA replication is the same.

Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

Tacrine therapy for the dementia of Alzheimer's disease.

Tacrine, a centrally acting cholinesterase inhibitor, may improve cognitive and functional status in patients with mild to moderate Alzheimer's disease. In recent controlled trials, patients have shown improvement in cognitive assessment scores, but the clinical significance of such benefits remains unclear. Appropriate diagnostic evaluation is necessary to prevent inappropriate treatment in patients with non-Alzheimer's dementia. Hepatotoxicity and gastrointestinal symptoms are common adverse effects of tacrine, and frequent monitoring of liver function is required.

Preferential formation and repair of chromium-induced DNA adducts and DNA--protein crosslinks in nuclear matrix DNA.

The distributions of chromium-DNA adducts and DNA-protein crosslinks induced by treatment of intact CHO cells with carcinogenic chromium were examined in distinct chromatin subfractions: a chromatin subfraction released by digestion of isolated nuclei with micrococcal nuclease (1SF, 14% of total nuclear DNA), bulk chromatin (74% of total DNA) and a nuclear matrix fraction (12% of total DNA). The identity of the matrix fraction was confirmed by hybridization of DNA from each subfraction with a cDNA probe prepared from total mRNA isolated from CHO cells, which showed that the 1SF and nuclear matrix fractions were 2.3- and 3.8-fold enriched in actively transcribed genes respectively, compared to total unfractionated DNA. Immediately following treatment of cells with 150 microM sodium chromate for 2 h the binding of chromium to each chromatin fraction was found to be non-uniform. Compared with total unfractionated nuclei, the nuclear matrix fractions were enriched in chromatin-bound chromium (3.4-fold), whereas the bulk chromatin fraction was relatively depleted (0.5-fold). Approximately 13% of nuclear chromium was associated with the detergent-soluble lipid component of nuclei. A similar distribution of chromatin-bound chromium was also apparent 24 h after the chromate treatment. Immediately after the 2 h chromate treatment, chromium-DNA adducts were detected in all the chromatin subfractions. Total nuclear and bulk chromatin DNA contained similar levels of this type of damage. The 1SF fraction was depleted approximately 3-fold in this type of damage compared with total nuclear DNA. In contrast, the nuclear matrix was markedly enriched in chromium-DNA adducts (approximately 4-fold compared with total nuclear DNA) at this time. As previously demonstrated, chromium-DNA adducts in total nuclear DNA decreased within the first 24 h, but thereafter persisted at a similar level. Chromium-DNA adducts in nuclear matrix DNA also reached maximum levels at the end of the 2 h treatment and decreased to 68% and 39% of this level by 24 and 48 h after treatment respectively. In contrast, the adduct levels in the 1SF and bulk chromatin fractions did not change up to 48 h after treatment. Chromium-induced DNA-protein crosslinks, which were stable to 8 M urea and 2% SDS, occurred almost exclusively in the nuclear matrix fraction. The crosslinks in this fraction reached a maximum level at the end of the 2 h treatment, but returned to control levels 24 h later.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoptosis is the mode of cell death caused by carcinogenic chromium.

The role of apoptosis in the mechanism of toxicity of hexavalent chromium, a human carcinogen, was investigated. Chinese hamster ovary (CHO) cells were treated with 150 or 300 microM sodium chromate for 2 hr, doses which decreased colony-forming efficiency to 53 and 5% of control, respectively. Cell growth was inhibited at least up to Day 8 after treatment. DNA synthesis was inhibited to 30 and 19% of control at 1 hr after treatment, and did not begin to recover until Day 4 after treatment. Protein synthesis was inhibited by 52 and 60% in 150 and 300 microM treated cells, respectively, 1 h after treatment, and recovered to 142 and 93%, respectively, at 24 hr. Incubation of cells with nontoxic doses of cycloheximide for 24 hr after treatment produced synergistic toxicity with chromate in colony-forming efficiency assays. Ion gradients persisted to Day 2 as revealed by exclusion of trypan blue dye in 97% of treated cells. Fluorescence microscopy of acridine orange-stained cells revealed morphological features of apoptosis including nuclear fragmentation in more than 90% of detached nonadherent cells and up to 22% of adherent cells by Day 2 after treatment. Untreated cells remained morphologically normal. Transmission electron microscopy of chromate treated cells showed characteristic features of apoptosis including chromatin margination and fragmentation, and cytoplasmic condensation with intact membrane and organelle structure. Internucleosomal DNA fragmentation (IDF) was delayed for at least 24 hr, whereafter it was detected in both adherent and nonadherent cells through Day 5 after treatment. These results indicate apoptosis as the mode of cell death caused by chromium and imply that apoptosis must be considered as a component of chromium-induced multistage carcinogenesis.

DNA polymerase arrest by adducted trivalent chromium.

Carcinogenic chromium (Cr6+) enters cells via the sulfate transport system and undergoes intracellular reduction to trivalent chromium, which strongly adducts to DNA. In this study, the effect of adducted trivalent chromium on in vitro DNA synthesis was analyzed with a polymerase-arrest assay in which prematurely terminated replication products were separated on a DNA sequencing gel. A synthetic DNA replication template was treated with increasing concentrations of chromium(III) chloride. The two lowest chromium doses used resulted in biologically relevant adduct levels (6 and 21 adducts per 1,000 DNA nucleotides) comparable with those measured in nuclear matrix DNA from cells treated with a 50% cytotoxic dose of sodium chromate in vivo. In vitro replication of the chromium-treated template DNA using the Sequenase version 2.0 T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH) resulted in dose-dependent polymerase arrest beginning at the lowest adduct levels analyzed. The pattern of polymerase arrest remained consistent as chromium adduct levels increased, with the most intense arrest sites occurring 1 base upstream of guanine residues on the template strand. Replication by the DNA polymerase I large (Klenow) fragment as well as by unmodified T7 DNA polymerase also resulted in similar chromium-induced polymerase arrest. Interstrand cross-linking between complementary strands was detected in template DNA containing 62, 111, and 223 chromium adducts per 1,000 DNA nucleotides but not in template containing 6 or 21 adducts per 1,000 DNA nucleotides, in which arrest nevertheless did occur. Low-level, dose-dependent interstrand cross-linking between primer and template DNA, however, was detectable even at the lowest chromium dose analyzed. Since only 9% of chromium adducts resulted in polymerase arrest in this system, we hypothesized that arrest occurred when the enzyme encountered chromium-mediated interstrand DNA-DNA cross-links between either the template and a separate DNA molecule or the template and its complementary strand in the same molecule. These results suggest that the obstruction of DNA replication by chromium-mediated DNA-DNA cross-links is a potential mechanism of chromium-induced genotoxicity in vivo.

Transcriptional inhibition by carcinogenic chromate: relationship to DNA damage.

Hexavalent chromium compounds are carcinogenic to humans, are potent inducers of tumors in experimental animals, and can neoplastically transform cells in culture. In this study, the effects of sodium chromate on the expression of the 78-kDa glucose-regulated protein (GRP78) gene and on general transcription were investigated with respect to the DNA damage induced by this agent. DNA single-strand breaks, DNA-protein cross-links, and chromium-DNA adducts were present in CHO cells immediately after 2 h of treatment with sodium chromate. Subsequently, these types of damage were repaired at different rates. Single-strand breaks were essentially repaired after 8 h. By 24 h posttreatment, no cross-links remained in cells exposed to 30 or 150 microM chromate, although cells treated with the 300-microM concentration still contained cross-links at that time. DNA-chromium adducts remained unrepaired for at least 32 h. The moderate constitutive level of GRP78 mRNA was not affected by chromate. Chromate did, however, suppress induction of this gene by tunicamycin in a concentration-and time-dependent manner. Thirty micromolar sodium chromate (96% survival), which caused the least DNA damage, had no effect on GRP78 induction, general RNA synthesis, or mRNA synthesis. Induction of GRP78 was suppressed immediately and 12 h after treatment with 150 microM chromate (54% survival), although there was a partial recovery of induction at 24 h after treatment, which correlated with the repair of DNA-protein cross-links. In contrast, both total cytoplasmic RNA and mRNA synthesis were suppressed by approximately 60-75% for at least 32 h by 150 microM chromate. At the 300-microM concentration (8% survival), where DNA-protein cross-links persisted beyond 24 h, GRP78 induction was totally suppressed for at least 24 h, while total RNA and mRNA synthesis were suppressed by 80-90% for at least 32 h. Overall, the effects of chromate on GRP78 induction correlated most closely with the presence of DNA-protein cross-links, but suppression of total RNA and mRNA synthesis correlated with the presence of DNA-chromium adducts. These results indicate that chromate exerts differential effects on the induction of the GRP78 gene and on general transcription.

Preoperative evaluation of the elderly patient.

Elderly patients are more likely to require surgery and to experience surgical complications than are younger patients. However, age should not be the sole factor in the decision for or against surgery. The surgical risk can be estimated on the basis of both age-related and disease-associated changes in the individual patient. Preoperative evaluation of the elderly patient requires assessment of cardiopulmonary and renal function, immune function, nutritional status and overall homeostatic capacity.