Clinical relevance of transgenic mouse models for aging research.

Studies on transgenic mice have shown them to be useful models for human aging- and age-related diseases. Life span end points in yeast and Caenorhabditis elegans can identify highly conserved genes that promote longevity when their functions are lost and which can readily be manipulated in the mouse. Protein kinase A is an example of a highly conserved gene that has age-delaying effects when specific subunits are suppressed or removed in the mouse, suggesting that loss of function may be a rational pharmacologic target. Gain of function is also an attractive clinical approach because expression levels of some vital genes may decrease in an age-related manner. The antioxidant enzyme catalase can delay aging when the human gene is inserted into mitochondria of mice. Other antioxidant genes are of interest in this system, both individually and in combination with catalase. A challenging aspect is to determine how to deliver catalase, as well as other gene products, into the mitochondria in the clinical setting. A number of new and exciting genes will most likely be investigated as clinical antiaging targets as the result of a forward genetic life span screening approach in invertebrates and a reverse genetic life span approach in the mouse.

Aberrant p21WAF1-dependent growth arrest as the possible mechanism of abnormal resistance to ultraviolet light cytotoxicity in Li-Fraumeni syndrome fibroblast strains heterozygous for TP53 mutations.

The purpose of this study is to better understand the roles of the p53 tumor suppressor protein and the product of the p53-regulated gene p21WAF1 in the response of diploid human dermal fibroblast cultures to 254 nm ultraviolet (UV) light. We report that Li-Fraumeni syndrome (LFS) fibroblast strains heterozygous for TP53 mutation at either codon 245 or 234 exhibit markedly reduced or no expression of p21WAF1 following UV irradiation, respectively. These strains also exhibit defective nucleotide excision repair and pronounced inhibition of RNA synthesis following UV exposure, both of which are molecular hallmarks of cells derived from patients with the UV-sensitive syndrome xeroderma pigmentosum. In sharp contrast to xeroderma pigmentosum cells, however, the repair-deficient LFS cells show abnormal resistance, rather than hypersensitivity, to the killing effect of UV light. We further demonstrate that exposure of normal human fibroblasts to biologically relevant fluences (< or = 15 J/m2) of UV does not induce apoptotic cell death, indicating that UV resistant phenotype displayed by LFS strains is not associated with deregulated apoptosis. In normal fibroblasts, such treatment results in a moderate ( threefold) up-regulation of p53 protein, induction of the p21WAF1 gene, and a senescence-like growth arrest. On the other hand, exposure to > or = 20 J/m2 UV results in a striking up-regulation of p53, inhibition of p21WAF1 expression, and activation of an apoptotic pathway. We conclude that: (i) p21WAF1-mediated senescence is the principal mode of cell death induced by < or = 15 J/m2 UV light in normal human fibroblasts; (ii) there is a threshold effect for p53-dependent apoptosis and that, in normal human cells, this threshold level is induced upon expsoure to 20 J/m2 UV; (iii) the p53 signaling pathway is malfunctional in the TP53 heterozygous LFS strains examined; and (iv) the enhanced resistance to UV-induced cell killing displayed by these LFS strains is a consequence of diminished growth arrest, which is presumably mediated by p21WAF1 and not abnormalities in an apoptotic pathway.

Characterization of the signal transduction pathway mediating gamma ray-induced inhibition of DNA synthesis in human cells: indirect evidence for involvement of calmodulin but not protein kinase C nor p53.

Cultured cells from patients inheriting the rare cancer-prone and radiotherapy-sensitive disorder ataxia-telangiectasia (A-T) exhibit anomalies in cell cycle control and protein kinase C (PKC)-mediated upregulation of p53 protein following exposure to ionizing radiation. It remains unclear, however, as to whether this irregularity in a p53-dependent signal transduction pathway controlling the G1/S checkpoint is causally linked to the most consistent molecular hallmark of A-T-namely, marked attenuation in the inhibition of replicative DNA synthesis at early times (< or = 2 h) after irradiation [radioresistant DNA synthesis (RDS)]. We report here that treatment of normal human fibroblast strains with inhibitors of calmodulin (CaM) (i.e. W7 and W13) and CaM-dependent protein kinases II and IV (i.e. KN62) prior to radiation exposure elicits an 'A-T-like' RDS phenotype, whereas treatment with PKC inhibitors (e.g. staurosporine) does not produce this response. Moreover, at 1 h post-gamma irradiation A-T fibroblasts undergo normal induction of p53 protein while exhibiting the RDS trait. At later times (e.g. 4 h) following irradiation, however, these A-T cells contain abnormally low levels of p53 protein, as do their lymphoblastoid cell line counterparts during the entire post-gamma ray incubation period. On the other hand, human cells which either lack the p53 gene completely (i.e. HL60 leukemia cells) or harbor a germline mutation in the gene (i.e. Li-Fraumeni syndrome cells) shut down their DNA replication machinery normally upon sustaining radiation damage. We thus conclude that the transitory delay in DNA synthesis routinely experienced by human cells in the face of radiation injury is mediated through a CaM-dependent regulatory cascade which involves neither PKC nor p53 protein. Accordingly, A-T cells appear to be malfunctional in at least two distinct radiation-responsive signalling pathways, one regulating the G1/S checkpoint and governed by p53 and PKC and another controlling passage through S phase and requiring CaM.

Effect of DNA polymerase inhibitors on repair of gamma ray-induced DNA damage in proliferating (intact versus permeable) human fibroblasts: evidence for differences in the modes of action of aphidicolin and 1-beta-D-arabinofuranosylcytosine.

The mammalian DNA polymerase inhibitors aphidicolin and 1-beta-D-arabinofuranosylcytosine (araC), when used in combination, inhibit the repair of DNA damage induced by gamma rays or 4-nitroquinoline 1-oxide in normal human fibroblasts to an extent 2- to 4-fold greater than that seen with each inhibitor alone. Thus either aphidicolin modulates the rate of intracellular accumulation of araC 5'-triphosphate (araCTP), the presumed rate-limiting step in the genotoxic action of araC, or aphidicolin and araC inhibit repair by different mechanisms. To explore these possibilities, we compared the effects of aphidicolin, araC, araCTP, and 2',3'-dideoxythymidine triphosphate (ddTTP) on repair of DNA damage induced by 60Co gamma radiation in intact versus permeable human fibroblasts. Both aphidicolin and araC strongly inhibited repair in permeable cells, as indicated by the accumulation of DNA strand breaks in irradiated cultures that were subsequently treated with saponin (25 micrograms/ml; 10 min) and incubated for 2 h with either chemical. The extent of repair inhibition by each drug was comparable in intact and permeable cells, amounting to approximately 1.1 sites/10(8) daltons/2 h upon exposure to 150 Gy. The active metabolite of araC, araCTP, did not inhibit repair in intact cells, but did so in permeable cells to an extent within the range of that seen with araC or aphidicolin alone. The incidence of DNA strand breaks accumulating in gamma-irradiated permeable cultures as a result of incubation with araCTP plus aphidicolin, or araC plus aphidicolin, was approximately 2-fold greater than that arising in parallel cultures which had been incubated with optimal concentrations of each of the three drugs alone. Although the resolution of our assays compelled us to monitor repair events in moribund cell populations, we have reason to be confident that within the short post-irradiation period considered here, the observed drug-accumulated breaks truly represent functional repair inhibition and not merely abortive pathological responses. We thus conclude that (1) the accumulation of araCTP in intact cells is not limiting the ability of araC to inhibit DNA repair; and (2) the mode of the inhibitory action of araC/araCTP on gamma ray repair is different from that of aphidicolin. In contrast to the observations with these chemicals, ddTTP (20 microM), a potent inhibitor of DNA polymerase beta, did not produce any measurable effect on DNA repair in gamma-irradiated permeable fibroblasts, nor did it enhance the efficacy of araC, araCTP or aphidicolin to inhibit repair. These results strongly suggest that DNA polymerase beta plays no significant role in the repair of gamma radioproducts in human fibroblasts.(ABSTRACT TRUNCATED AT 400 WORDS)

Association of ATM activation and DNA repair with induced radioresistance after low-dose irradiation.

Mammalian cells often exhibit a hyper-radiosensitivity (HRS) to radiation doses <20 cGy, followed by increased radioresistance (IRR) at slightly higher doses (∼20-30 cGy). Here, the influence of DNA double-strand break repair (DSBR) on IRR was examined. The failure of Ataxia telangiectasia (AT) cells to undergo IRR reported by others was confirmed. Flow cytometric analysis indicated that normal cells fail to show a measurable increase in serine 1981 phosphorylated AT-mutated (ATM) protein after 10 cGy up to 4 h post irradiation, but a two- to fourfold increase after 25 cGy. Similarly, more proficient reduction of phosphorylated histone H2AX was observed 24 h after 25 cGy than after 10 cGy, suggesting that DSBR is more efficient during IRR than HRS. A direct examination of the consequences of inefficient DNA repair per se (as opposed to ATM-mediated signal transduction/cell cycle responses), by determining the clonogenic survival of cells lacking the DNA repair enzyme polynucleotide kinase/phosphatase, indicated that these cells have a response similar to AT cells, i.e. HRS but no IRR, strongly linking IRR to DSBR.

Role of gestational hormones in the induction of insulin resistance.

Pregnancy is associated with insulin resistance. We studied insulin binding and postbinding function in isolated adipocytes from pregnant and nonpregnant rats. We also used a primary culture system for female virgin rat adipocytes to assess the effects of gestational hormones in vitro on insulin binding and postbinding function. Insulin binding to adipocytes was normal during pregnancy, but [14C]3-O-methylglucose transport was reduced. When hCG or estradiol was added to the culture medium, no change in maximum [14C]3-O-methylglucose transport was found; however, maximum insulin binding was increased with estradiol. Progesterone and cortisol both decreased maximum insulin binding and [14C]3-O-methylglucose transport. PRL and placental lactogen decreased maximum [14C]3-O-methylglucose transport, but did not change insulin binding. When these hormones were added concurrently no change in insulin binding was found, but maximum [14C]3-O-methylglucose transport was reduced. We conclude that the insulin resistance of pregnancy is associated with a postbinding defect in insulin action. Estradiol increased insulin receptor binding, but during pregnancy this effect may be offset by the reduction in insulin binding induced by progesterone and cortisol. The postbinding defect in insulin action during pregnancy is probably related to increasing amounts of progesterone, cortisol, PRL, and placental lactogen.

A study of gamma-aminobutyric acid uptake in normal and Down's syndrome platelets.

1. Initial uptake rates of gamma-aminobutyric acid (GABA) were compared in Down's syndrome (D.S.) and normal platelets GABA uptake was decreased in D.S. platelets (1.10 +/- 0.10 nmol h-1 10(-9)) compared to uptake by normal platelets (1.89 +/- 0.21 nmol h-1 10(-9), P less than 0.005. 2. The effect of varying the NA+ concentration was similar on D.S. and normal platelets. Increasing the media Na+ concentration resulted in increased rates of GABA uptake in both D.S. and normal platelets. 3. GABA uptake in the presence of 2,4-dinitrophenol or at 2 degrees C is approximately 56% of the uptake at 37 degrees C for both D.S. and normal platelets. 4. Extrapolation of a reciprocal plot indicates a two affinity uptake system; a high affinity and a low affinity mechanism. 5. A significant defect in GABA uptake exists in D.S. platelets.

Inhibition of DNA synthesis and G1/S-phase transition in normal human fibroblasts elicited by a heat-labile trans-acting factor in gamma-irradiated HeLa cell extracts.

Proliferating human cells exposed to ionizing radiation show complex cellular responses including a delay in progression through various phases in the cell cycle. These cell cycle checkpoints are regulated by mitogenic signaling pathways which transduce the extracellular signals to the cell cycle control machinery. In this study we demonstrate that microinjection of a cellular extract, prepared from gamma-irradiated (40 Gy) HeLa cells, into the cytoplasm of normal human fibroblasts results in suppression of DNA replicative synthesis, indicating the presence of a trans-acting DNA synthesis-inhibiting factor(s). The addition of this same extract to the culture medium for a short time (< or = 2 h) also inhibits DNA synthesis in human fibroblasts, affecting both replicon initiation and DNA chain elongation processes. Moreover, a 2-h incubation of the fibroblast cultures with the extract causes a transient delay in cell progression from G1 to S phase coupled with up-regulation of the p53 tumor suppressor protein. Both the DNA synthesis-inhibiting and G1-phase-blocking activities are reduced markedly when the extract is heated (80 degrees C; 10 min) prior to its addition to the culture medium. On the other hand, pretreatment of the fibroblast cultures with KN62, an inhibitor of calmodulin-dependent kinase II (CaMKII), serves to abrogate the inhibitory effect of the extract on DNA synthesis without influencing its ability to induce the G1-phase block. These results are compatible with the presence in HeLa cell extracts of a heat-labile trans-acting factor that triggers, in normal human cells, the activation of (1) a CaMKII-dependent signal transduction pathway mediating suppression of DNA synthesis and (2) a p53-dependent pathway mediating G1-phase checkpoint control.

Radiosensitivity in ataxia telangiectasia fibroblasts is not associated with deregulated apoptosis.

Ataxia telangiectasia (AT) is an autosomal recessive human disorder featuring diverse clinical abnormalities including proneness to cancer and extreme sensitivity to ionizing radiation. Although cells from AT patients exhibit faulty activation of the p53 signal transduction pathway at early times after radiation exposure, it has been proposed that high levels of DNA damage persisting in AT cells may up-regulate p53 through an ATM-independent mechanism at late times after irradiation, leading to cell death by apoptosis. In this study we demonstrate that diploid skin fibroblast strains homozygous for the AT mutation fail to up-regulate p53 protein at late times (< or = 48 h) after irradiation with 60Co gamma rays. Moreover, exposure of normal and AT fibroblasts to a dose of 8 Gy does not result in a significant increase in the fraction of apoptotic cells. Since this treatment reduces the clonogenic potential of human cells by at least two orders of magnitude, we conclude that apoptosis is not the primary mechanism of cell death induced by ionizing radiation in human normal and AT fibroblast cultures. Therefore, our results are not in accordance with the current hypothesis suggesting that increased radiosensitivity of AT cells is associated with deregulated apoptosis.

Lack of correlation between DNA strand breakage and p53 protein levels in human fibroblast strains exposed to ultraviolet lights.

The contribution of DNA strand breaks accumulating in the course of nucleotide excision repair to upregulation of the p53 tumor suppressor protein was investigated in human dermal fibroblast strains after treatment with 254 nm ultraviolet (UV) light. For this purpose, fibroblast cultures were exposed to UV and incubated for 3 h in the presence or absence of l-beta-D-arabinofuranosylcytosine (araC) and/or hydroxyurea (HU), and then assayed for DNA strand breakage and p53 protein levels. As expected from previous studies, incubation of normal and ataxia telangiectasia (AT) fibroblasts with araC and HU after UV irradiation resulted in an accumulation of DNA strand breaks. Such araC/HU-accumulated strand breaks (reflecting nonligated repair-incision events) following UV irradiation were not detected in xeroderma pigmentosum (XP) fibroblast strains belonging to complementation groups A and G. Western blot analysis revealed that normal fibroblasts exhibited little upregulation of p53 (approximately 1.2-fold) when incubated without araC after 5 J/m2 irradiation, but showed significant (three-fold) upregulation of p53 when incubated with araC after irradiation. AraC is known to inhibit nucleotide excision repair at both the damage removal and repair resynthesis steps. Therefore, the potentiation of UV-induced upregulation of p53 evoked by araC in normal cells may be a consequence of either persistent bulky DNA lesions or persistent incision-associated DNA strand breaks. To distinguish between these two possibilities, we determined p53 induction in AT fibroblasts (which do not upregulate p53 in response to DNA strand breakage) and in XP fibroblasts (which do not exhibit incision-associated breaks after UV irradiation). The p53 response after treatment with 5 J/m2 UV and incubation with araC was similar in AT, XPA, XPG and normal fibroblasts. In addition, exposure of XPA and XPG fibroblasts to UV (5, 10 or 20 J/m2) followed by incubation without araC resulted in a strong upregulation of p53. We further demonstrated that HU, an inhibitor of replicative DNA synthesis (but not of nucleotide excision repair), had no significant impact on p53 protein levels in UV irradiated and unirradiated human fibroblasts. We conclude that upregulation of p53 at early times after exposure of diploid human fibroblasts to UV light is triggered by persistent bulky DNA lesions, and that incision-associated DNA strand breaks accumulating in the course of nucleotide excision repair and breaks arising as a result of inhibition of DNA replication contribute little (if anything) to upregulation of p53.

Gel microdrop flow cytometry assay for low-dose studies of chemical and radiation cytotoxicity.

Low-level cytotoxicity may affect low-dose dose-response relations for cancer and other endpoints. Conventional colony-forming assays are rarely sensitive enough to examine small changes in cell survival and growth. Automated image-analysis techniques are limited to ca. 10(4) cells/plate. An alternative method involves encapsulation of single proliferating cells into ca. 35-75-microm-diameter agarose gel microdrops (GMDs) that are randomly grouped, differential exposure of these groups, culture at 37 degrees C for 3-5 days, and finally GMD analysis by flow cytometry (FC) to determine the ratio of GMDs containing multiple versus single cells as a measure of clonogenic survival. This GMD/FC assay was used to examine low-dose cell killing induced by a cooked-meat mutagen/rodent-carcinogen (MeIQx) in DNA-repair-deficient/metabolically-sensitive CHO cells. Results of conventional colony-forming assays using up to 30 replicate plates indicate a shouldered, threshold-like dose-response; in contrast, those obtained using the GMD/FC assay suggest "hypersensitivity"-like nonlinearity in dose-response. The GMD/FC assay was also applied to human A549 lung cells after GMD-encapsulation and gamma radiation followed by culture for a total of 4 days, to examine survival after exposure to > or =100 cGy delivered at a relatively low dose rate (0.18 cGy/min). Dose-response for clonogenic growth was again observed to be reduced with apparent nonlinear suggesting hypersensitivity between 0 and 50 cGy, insofar as doses of 5 and 10 cGy appear to be ca. fivefold more effective per unit dose than the 50- or 100-cGy doses used. The GMD/FC assay may thus reveal low-dose dose-response relations for chemical and radiation effects on cell proliferation/killing with implications for low-dose risk assessment.

Self-policing: it's our duty.

As professionals, dental hygienists have a duty to protect the public from incompetent and unethical behaviour. This literature review was completed to provide a guide for dental hygienists who may not feel confident addressing errant behaviour. It discusses principles of biomedical ethics and uses Morriem's system of evaluating levels of adverse outcome as an assessment tool. Pertinent examples in dental hygiene practice are included. Investigation and resolution of dilemmas are also discussed with the aim of upholding the integrity of dental hygiene practice.

Nucleotide sequence of 5 S ribosomal ribonucleic acid of Iguana iguana.

Cell cultures of the reptile Iguana iguana have been labeled with 32PO4(3-) and the resulting radioactive 5 S rRNA isolated. Enzymatic digests of the radioactive RNA were fractionated by standard procedures, and the products of the digestions sequenced. From the sequences of the oligonucleotides produced by enzymatic digestion of the Iguana 5 S rRNA it is possible to propose a primary sequence for this RNA which differs in only two positions (2 and 25) from the sequence known for mammalian 5 S rRNAs. This sequence is closer to that of mammals than is that of any other nonmammalian species yet examined.

Effects of the protein kinase inhibitors wortmannin and KN62 on cellular radiosensitivity and radiation-activated S phase and G1/S checkpoints in normal human fibroblasts.

Wortmannin is a potent inhibitor of phosphatidylinositol (PI) 3-kinase and PI 3-kinase-related proteins (e.g. ATM), but it does not inhibit the activity of purified calmodulin-dependent protein kinase II (CaMKII). In the present study, we compared the effects of wortmannin and the CaMKII inhibitor KN62 on the response of normal human dermal fibroblast cultures to gamma radiation. We demonstrate that wortmannin confers a phenotype on normal fibroblasts remarkably similar to that characteristic of cells homozygous for the ATM mutation. Thus wortmannin-treated normal fibroblasts exhibit increased sensitivity to radiation-induced cell killing, lack of temporary block in transition from G1 to S phase following irradiation (i.e. impaired G1/S checkpoint), and radioresistant DNA synthesis (i.e. impaired S phase checkpoint). Wortmannin-treated cultures display a diminished capacity for radiation-induced up-regulation of p53 protein and expression of p21WAF1, a p53-regulated gene involved in cell cycle arrest at the G1/S border; the treated cultures also exhibit decreased capacity for enhancement of CaMKII activity post-irradiation, known to be necessary for triggering the S phase checkpoint. We further demonstrate that KN62 confers a radioresistant DNA synthesis phenotype on normal fibroblasts and moderately potentiates their sensitivity to killing by gamma rays, without modulating G1/S checkpoint, p53 up-regulation and p21WAF1 expression following radiation exposure. We conclude that CaMKII is involved in the radiation responsive signalling pathway mediating S phase checkpoint but not in the p53-dependent pathway controlling G1/S checkpoint, and that a wortmannin-sensitive kinase functions upstream in both pathways.

Faulty DNA polymerase delta/epsilon-mediated excision repair in response to gamma radiation or ultraviolet light in p53-deficient fibroblast strains from affected members of a cancer-prone family with Li-Fraumeni syndrome.

Dermal fibroblast strains cultured from affected members of a cancer-prone family with Li-Fraumeni syndrome (LFS) harbor a point mutation in one allele of the p53 tumor suppressor gene, resulting in loss of normal p53 function. In this study we have examined the ability of these p53-deficient strains to carry out the long-patch mode of excision repair, mediated by DNA polymerases delta and epsilon, after exposure to 60Co gamma radiation or far ultraviolet (UV) (chiefly 254 nm) light. Repair was monitored by incubation of the irradiated cultures in the presence of aphidicolin (apc) or 1-beta-D-arabinofuranosylcytosine (araC), each a specific inhibitor of long-patch repair, followed by measurement of drug-induced DNA strand breaks (reflecting non-ligated strand incision events) by alkaline sucrose velocity sedimentation. The LFS strains displayed deficient repair capacity in response to both gamma rays and UV light. The repair anomaly in UV-irradiated LFS cultures was manifested not only in the overall genome, but also in the transcriptionally active, preferentially repaired c-myc gene. Using autoradiography we also assessed unscheduled DNA synthesis (UDS) after UV irradiation and found this conventional measure of repair replication to be deficient in LFS strains. Moreover, both apc and araC decreased the level of UV-induced UDS by approximately 75% in normal cells, but each had only a marginal effect on LFS cells. We further demonstrated that the LFS strains are impaired in the recovery of both RNA and replicative DNA syntheses after UV treatment, two molecular anomalies of the DNA repair deficiency disorders xeroderma pigmentosum and Cockayne's syndrome. Together these results imply a critical role for wild-type p53 protein in DNA polymerase delta/epsilon-mediated excision repair, both the mechanism operating on the entire genome and that acting on expressed genes.

Comparative genotoxicity of 2,3'-O-cyclocytidine, beta-xylocytidine and 1-beta-D-arabinofuranosylcytosine in human tumor cell lines.

We have investigated the genotoxicity of two 3'-derivatives of cytidine, 2,3'-O-cyclocytidine (3'-cycloC) and beta-xylocytidine (xyloC), in human leukemia and solid tumor cell lines. Both derivatives were found to be cytotoxic at micromolar concentrations. For example, in the alveolar tumor cell line A549 which was included in all experiments as a reference, drug concentrations required to induce 50% inhibition of cell growth (D50 values) equalled 55 microM for 3'-cycloC and 80 microM for xyloC. Compared with the response of this reference cell line, none of the solid tumor cell lines tested--representing five different malignancies--displayed significant hypersensitivity to these drugs, while the acute lymphoblastic leukemia cell lines proved to be hypersensitive (range of D50 values, 5-13 microM). To gain insight into the modes of cytotoxic action of xyloC and 3'-cycloC, we compared the effect on DNA metabolism of these compounds with that of 1-beta-D-arabinofuranosylcytosine (araC), a potent inhibitor of semi-conservative DNA replication and long-patch excision repair. As seen with araC, the xylo compound strongly inhibited both DNA replicative synthesis and the repair of DNA damage induced by UV light and 60Co gamma-radiation. In gamma-irradiated A549 cells, the extent of repair inhibition by 1 mM xyloC was approximately 40% of that inhibited by araC, and concomitant exposure of the irradiated cultures to xyloC plus araC gave rise to a synergistic response. Since araC was employed at a concentration (0.1 mM) which produced a maximal effect on DNA repair when applied alone, the observed synergistic response implies that the mode of action of xyloC on DNA repair is different from that of araC. In contrast to that observed with xyloC, 3'-cycloC proved to be a very weak inhibitor of DNA replication and repair, strongly suggesting that the genotoxic action of the latter analog may be through a mechanism other than inhibition of DNA synthesis.