Ethnoracial Disparities in Rates of Non-Natural Causes of Death After the 2020 COVID-19 Outbreak in New York State.

Introduction: COVID-19 was associated with increases in non-natural cause mortality in the U.S., including deaths due to drug overdose, homicide, and motor vehicle crashes. Initial reports indicated higher rates of non-natural mortality among ethnoracial minority groups. This report aims to clarify these disparities by documenting trends in non-natural mortality across ethnoracial groups during the 2020 COVID-19 surge in New York State. Methods: We report monthly trends in non-natural cause mortality (overall and stratified by ethnoracial status) in New York State from January 2019 through December 2020, which included the COVID-19 onset in March 2020. Results: Total mean monthly unintentional overdose rates per 100,000 increased from 17.45 (before surge: January 2019-February 2020) to 23.19 (after surge: March 2020-December 2020) (mean difference=5.73, 95% CI=3.82, 7.65; p<0.001). Mean monthly homicide death rates increased from 2.34 before surge to 3.55 after surge (mean difference=1.20, 95% CI=0.60, 1.81; p<0.001), with the increase seen primarily in the non-Latinx Black population. Although increasing unintentional overdose death rates before surge equally affected non-Latinx White, Latinx, and non-Latinx Black persons, they remained high for non-Latinx Black persons but dropped for the other 2 groups after the pandemic onset. None of the ethnoracial subgroups showed significant increases in suicide or motor vehicle crash death rates. Conclusions: Non-Latinx Black persons showed disproportionately high and sustained increased rates of unintentional overdose and homicide death rates after the 2020 COVID-19 surge in New York State. Fatality review and death scene investigation research is needed to better understand these disparities.

Elevated dNTP levels suppress hyper-recombination in Saccharomyces cerevisiae S-phase checkpoint mutants.

MEC1, the essential yeast homolog of the human ATR/ATM genes, controls the S-phase checkpoint and prevents replication fork collapse at slow zones of DNA replication. The viability of hypomorphic mec1-21 is reduced in the rad52 mutant, defective in homologous recombination, suggesting that replication generates recombinogenic lesions. We previously observed a 6-, 10- and 30-fold higher rate of spontaneous sister chromatid exchange (SCE), heteroallelic recombination and translocations, respectively, in mec1-21 mutants compared to wild-type. Here we report that the hyper-recombination phenotype correlates with lower deoxyribonucleoside triphosphate (dNTP) levels, compared to wild-type. By introducing a dun1 mutation, thus eliminating inducible expression of ribonucleotide reductase in mec1-21, rates of spontaneous SCE increased 15-fold above wild-type. All the hyper-recombination phenotypes were reduced by SML1 deletions, which increase dNTP levels. Measurements of dNTP pools indicated that, compared to wild-type, there was a significant decrease in dNTP levels in mec1-21, dun1 and mec1-21 dun1, while the dNTP levels of mec1-21 sml1, mec1-21 dun1 sml1 and sml1 mutants were approximately 2-fold higher. Interestingly, higher dNTP levels in mec1-21 dun1 sml1 correlate with approximately 2-fold higher rate of spontaneous mutagenesis, compared to mec1-21 dun1. We suggest that higher dNTP levels in specific checkpoint mutants suppress the formation of recombinogenic lesions.

Long-term follow-up of children with confirmed newborn screening disorders using record linkage.

BACKGROUND: Long-term follow-up of children identified through newborn screening is a critical process of data collection and analysis for advancing the public health understanding of the health outcomes and service uptake of the affected children. This article describes first steps toward the long-term follow-up of newborn screening children with confirmed disorders through records linkage using population-based administrative data. METHODS: The study cohort consisted of children born in 2006-2007 with confirmed disorders identified through newborn screening. Deterministic data linkage methods were used for record matching. RESULTS: The cohort was followed up to 2 years after birth by matching to data sources including vital records, hospital discharges, the Congenital Malformations Registry, and Early Intervention to monitor service utilization, comorbidities, and mortality of the affected children. Of 1215 children with confirmed conditions identified through newborn screening, 25 deaths (2.1%) were identified, 86.1% used hospital (in- or outpatient) services, 36.1% were enrolled in the Congenital Malformations Registry, and 19.9% used the services of the Early Intervention program during the 2-year follow-up period. CONCLUSIONS: Long-term follow-up of children with disorders identified through newborn screening can be initiated by using existing administrative data. This method is an inexpensive, cost-effective. and efficient approach for periodical assessment of services utilization, the efficiency of service delivery, and health outcomes for affected individuals.

Resveratrol is pro-apoptotic and thyroid hormone is anti-apoptotic in glioma cells: both actions are integrin and ERK mediated.

The stilbene resveratrol (RV) initiates p53-dependent apoptosis via plasma membrane integrin alphaVbeta3 in human cancer cells. A thyroid hormone (L-thyroxine, T(4)) membrane receptor also exists on alphaVbeta3. Stilbene and T(4) signals are both transduced by extracellular-regulated kinases 1 and 2 (ERK1/2); however, T(4) promotes cell proliferation in cancer cells, whereas RV is pro-apoptotic. Thyroid hormone has been shown to interfere with RV-induced apoptosis. However, the mechanisms involved are not fully understood. In this study, we examined the mechanism whereby T(4) inhibits RV-induced apoptosis in glioma cells. RV activated conventional protein kinase C and ERK1/2 and caused nuclear localization of cyclooxygenase-2 (COX-2), consequent p53 phosphorylation and apoptosis. RV-induced ERK1/2 activation is involved in not only COX-2 expression but also nuclear COX-2 accumulation. NS-398, a COX-2 inhibitor, did not affect ERK1/2 activation, but reduced the nuclear abundance of COX-2 protein and the formation of complexes of nuclear COX-2 and activated ERK1/2 that are required for p53-dependent apoptosis in RV-treated cells. T(4) inhibited RV-induced nuclear COX-2 and cytosolic pro-apoptotic protein, BcLx-s, accumulation. Furthermore, T(4) inhibited RV-induced apoptosis by interfering with the interaction of nuclear COX-2 and ERK1/2. This effect of T(4) was prevented by tetraiodothyroacetic acid (tetrac), an inhibitor of the binding of thyroid hormone to its integrin receptor. Tetrac did not, in the absence of T(4), affect induction of apoptosis by RV. Thus, the receptor sites on alphaVbeta3 for RV and thyroid hormone are discrete and activate ERK1/2-dependent downstream effects on apoptosis that are distinctive.

Resveratrol causes COX-2- and p53-dependent apoptosis in head and neck squamous cell cancer cells.

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.

Stimulation of sister chromatid exchanges and mutation by aflatoxin B1-DNA adducts in Saccharomyces cerevisiae requires MEC1 (ATR), RAD53, and DUN1.

The hepatocarcinogen aflatoxin B(1) (AFB(1)) is a potent recombinagen but weak mutagen in the yeast Saccharomyces cerevisiae. AFB(1) exposure induces DNA damage-inducible genes, such as RAD51 and those encoding ribonucleotide reductase (RNR), through a MEC1 (ATR homolog)-dependent pathway. Previous studies have indicated that MEC1 is required for both AFB(1)-associated recombination and mutation, and suggested that AFB(1)-DNA adducts are common substrates for recombination and mutagenesis. However, little is known about the downstream effectors of MEC1 required for genotoxic events associated with AFB(1) exposure. Here we show that AFB(1) exposure increases frequencies of RAD51-dependent unequal sister chromatid exchange (SCE) and activates Rad53 (CHK2). We found that MEC1, RAD53, and DUN1 are required for both AFB(1)-associated mutation and SCE. Deletion of SML1, which encodes an inhibitor of RNR, did not suppress the DUN1-dependent requirement for AFB(1)-associated genetic events, indicating that higher dNTP levels could not suppress the dun1 phenotype. We identified AFB(1)-DNA adducts and show that approximately the same number of adducts are obtained in both wild type and rad53 mutants. Since DUN1 is not required for UV-associated mutation and recombination, these studies define a distinct role for DUN1 in AFB(1)-associated mutagenesis and recombination. We speculate that AFB(1)-associated DNA adducts stall DNA replication, a consequence of which can either be mutation or recombination.

UV but not X rays stimulate homologous recombination between sister chromatids and homologs in a Saccharomyces cerevisiae mec1 (ATR) hypomorphic mutant.

MEC1, the essential yeast ATM/ATR homolog, prevents replication fork collapse and is required for the cellular response to DNA damage. We had previously observed higher rates of spontaneous SCE, heteroallelic recombination and translocations in mec1-21 mutants, which still retain some G2 checkpoint function, compared to mec1 null mutants, which are completely defective in checkpoint function, and wild type. However, the types of DNA lesions that are more recombinogenic in mec1-21, compared to wild type, are unknown. Here, we measured DNA damage-associated SCE, homolog (heteroallelic) recombination, and homology-directed translocations in mec1-21, and characterized types of DNA damage-associated chromosomal rearrangements that occur in mec1-21. Although frequencies of UV-associated recombination were higher in mec1-21, the mutant was defective in double-strand break-associated SCE and heteroallelic recombination. Over-expression of Rad53 in mec1-21 reduced UV-associated recombination but did not suppress the defect in X-ray-associated recombination. Both X ray and UV exposure increased translocation frequencies in mec1-21, but the majority of the UV-associated products were non-reciprocal translocations. We suggest that although recombinational repair of double-stand breaks is less efficient in mec1 mutants, recombinants may be generated by other mechanisms, such as break-induced replication.

Factors associated with 1-year mortality after discharge for acute stroke: what matters?

OBJECTIVE: To evaluate factors associated with 1-year mortality after discharge for acute stroke. METHODS: In this retrospective cohort study, we studied 305 patients with ischemic stroke or intracerebral hemorrhage discharged in 2010/2011. We linked Get With The Guidelines®-Stroke clinical data with New York State administrative data and used multivariate regression models to examine variables related to 1-year all-cause mortality poststroke. RESULTS: The mean age was 68.6 ± 14.8 years and 51.1% were women. A total of 146 (47.9%) were discharged directly home, 96 (31.5%) to inpatient rehabilitation facilities (IRFs), and 63 (20.7%) to skilled nursing facilities (SNFs). Overall, 24 (7.9%) patients died within 1-year post-discharge. Older age (adjusted odds ratio [OR] 1.05, 95% confidence interval [CI] 1.00-1.10), higher National Institutes of Health Stroke Scale (NIHSS) on admission (OR 1.10, 95% CI 1.03-1.17), and discharge destination (IRF vs. home, OR 0.10, 95% CI 0.01-0.94; and SNF vs. home, OR 2.22, 95% CI 0.71-6.95) were factors associated with 1-year all-cause mortality. When ambulation status at discharge was added to the model, ambulation with assistance and non-ambulation were significantly associated with mortality (ambulatory with assistance vs. ambulatory, OR 9.42, 95% CI 1.87-47.61; nonambulatory vs. ambulatory, OR 12.65, 95% CI 1.89-84.89). CONCLUSIONS: While age and NIHSS on admission are important predictors of long-term outcomes, factors at discharge - ambulation status at discharge and discharge destination - are associated with 1-year mortality post-discharge for acute stroke and therefore could represent therapeutic targets to improve long-term survival in future studies.

Transcriptional response of yeast to aflatoxin B1: recombinational repair involving RAD51 and RAD1.

The potent carcinogen aflatoxin B(1) is a weak mutagen but a strong recombinagen in Saccharomyces cerevisiae. Aflatoxin B(1) exposure greatly increases frequencies of both heteroallelic recombination and chromosomal translocations. We analyzed the gene expression pattern of diploid cells exposed to aflatoxin B(1) using high-density oligonucleotide arrays comprising specific probes for all 6218 open reading frames. Among 183 responsive genes, 46 are involved in either DNA repair or in control of cell growth and division. Inducible growth control genes include those in the TOR signaling pathway and SPO12, whereas PKC1 is downregulated. Eleven of the 15 inducible DNA repair genes, including RAD51, participate in recombination. Survival and translocation frequencies are reduced in the rad51 diploid after aflatoxin B(1) exposure. In mec1 checkpoint mutants, aflatoxin B(1) exposure does not induce RAD51 expression or increase translocation frequencies; however, when RAD51 is constitutively overexpressed in the mec1 mutant, aflatoxin B(1) exposure increased translocation frequencies. Thus the transcriptional profile after aflatoxin B(1) exposure may elucidate the genotoxic properties of aflatoxin B(1).

Both RAD5-dependent and independent pathways are involved in DNA damage-associated sister chromatid exchange in budding yeast.

Sister chromatids are preferred substrates for recombinational repair after cells are exposed to DNA damage. While some agents directly cause double-strand breaks (DSBs), others form DNA base adducts which stall or impede the DNA replication fork. We asked which types of DNA damage can stimulate SCE in budding yeast mutants defective in template switch mechanisms and whether PCNA polyubiquitination functions are required for DNA damage-associated SCE after exposure to potent recombinagens. We measured spontaneous and DNA damage-associated unequal sister chromatid exchange (uSCE) in yeast strains containing two fragments of his3 after exposure to MMS, 4-NQO, UV, X rays, and HO endonuclease-induced DSBs. We determined whether other genes in the pathway for template switching, including UBC13, MMS2, SGS1, and SRS2 were required for DNA damage-associated SCE. RAD5 was required for DNA damage-associated SCE after exposure to UV, MMS, and 4-NQO, but not for spontaneous, X-ray-associated, or HO endonuclease-induced SCE. While UBC13, MMS2, and SGS1 were required for MMS and 4NQO-associated SCE, they were not required for UV-associated SCE. DNA damage-associated recombination between his3 recombination substrates on non-homologous recombination was enhanced in rad5 mutants. These results demonstrate that DNA damaging agents that cause DSBs stimulate SCE by RAD5-independent mechanisms, while several potent agents that generate bulky DNA adducts stimulate SCE by multiple RAD5-dependent mechanisms. We suggest that DSB-associated recombination that occurs in G2 is RAD5-independent.

Saccharomyces cerevisiae RAD53 (CHK2) but not CHK1 is required for double-strand break-initiated SCE and DNA damage-associated SCE after exposure to X rays and chemical agents.

Saccharomyces cerevisiae RAD53 (CHK2) and CHK1 control two parallel branches of the RAD9-mediated pathway for DNA damage-induced G(2) arrest. Previous studies indicate that RAD9 is required for X-ray-associated sister chromatid exchange (SCE), suppresses homology-directed translocations, and is involved in pathways for double-strand break repair (DSB) repair that are different than those controlled by PDS1. We measured DNA damage-associated SCE in strains containing two tandem fragments of his3, his3-Delta5' and his3-Delta3'::HOcs, and rates of spontaneous translocations in diploids containing GAL1::his3-Delta5' and trp1::his3-Delta3'::HOcs. DNA damage-associated SCE was measured after log phase cells were exposed to methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4-NQO), UV, X rays and HO-induced DSBs. We observed that rad53 mutants were defective in MMS-, 4-NQO, X-ray-associated and HO-induced SCE but not in UV-associated SCE. Similar to rad9 pds1 double mutants, rad53 pds1 double mutants exhibited more X-ray sensitivity than the single mutants. rad53 sml1 diploid mutants exhibited a 10-fold higher rate of spontaneous translocations compared to the sml1 diploid mutants. chk1 mutants were not deficient in DNA damage-associated SCE after exposure to DNA damaging agents or after DSBs were generated at trp1::his3-Delta5'his3-Delta3'::HOcs. These data indicate that RAD53, not CHK1, is required for DSB-initiated SCE, and DNA damage-associated SCE after exposure to X-ray-mimetic and UV-mimetic chemicals.

Did summer weather factors affect gastrointestinal infection hospitalizations in New York State?

BACKGROUND: Gastrointestinal infections, a substantial public health problem worldwide, have been associated with single weather factors but the joint effect of multiple weather factors on gastrointestinal infections remains unclear. METHODS: We conducted a retrospective time-series analysis to investigate effects of weather conditions on hospitalizations for gastrointestinal infections (GIH) in New York State in July and August from 1991 to 2004. Applying generalized additive model (GAM), we evaluated the associations between daily GIH count and multiple weather factors including temperature, humidity, and precipitation (0-10 lag days), adjusting for long term trend, seasonality, and calendar effects. RESULTS: Maximum temperature, minimum temperature, and maximum universal apparent temperature (UAT) showed that each °C increase in temperature was significantly associated with an increase (0.70-0.96%) in daily GIH count, with the greatest impacts observed at lag 1. Extreme heat (EH: >90th percentile) (3.68% at lag 1) and precipitation (0.31% at lag 4) showed larger impacts on increases of GIH and a clear dose-response relationship for EH. Stratified analyses showed that the impacts of extreme heat on GIH tend to be greater among Hispanics, blacks, females, and those with bacterial infections. CONCLUSION: We found that high maximum and minimum temperature, UAT, precipitation, and extreme heat in summer significantly increased the risks of GIH in NYS. Our findings also suggest that bacteria might be a significant cause for GIH in the summer, and minority, female and those with bacterial infection may be more vulnerable to heat's effects on GIH.

Effect of Hurricane Sandy on Health Care Services Utilization Under Medicaid.

OBJECTIVE: This investigation assessed changes in utilization of inpatient, outpatient, emergency department, and pharmacy services in the aftermath of Hurricane Sandy in 8 counties in New York affected by the storm. METHODS: Medicaid data for enrollees residing in 8 counties in New York were used to obtain aggregated daily counts of claims for 4 service types over immediate, 3-month, and 1-year periods following the storm. Negative binomial regression was used to compare service utilization in the storm year with the 2 prior years, within areas differentially affected by the storm. RESULTS: Changes in service utilization within areas inside or outside the storm zone were most pronounced over the 1-year effect period. Differences in service utilization by year were the same by storm zone designation over the immediate effect period for all services. CONCLUSIONS: Results are consistent with previous investigations demonstrating that some of the greatest effects of a disaster on health services utilization occur well beyond the initial event. One-year effects, combined with some 3-month effects, suggests that storm recovery, with its effect on health care services utilization, may have followed different paths in areas designated as inside or outside the storm zone. (Disaster Med Public Health Preparedness. 2016;10:472-484).

Androgen-induced human breast cancer cell proliferation is mediated by discrete mechanisms in estrogen receptor-alpha-positive and -negative breast cancer cells.

Androgens have important physiological effects in women. Not only are they the precursor hormones for estrogen biosynthesis in the ovaries and extragonadal tissues, but also act directly via androgen receptors (ARs) throughout the body. Studies of the role of androgens on breast cancer development are controversial and the mechanisms involved are not fully understood. In this report we demonstrate that a non-aromatizable androgen metabolite, dihydrotestosterone (DHT), stimulated cell proliferation in vitro of both estrogen receptor-alpha (ER-alpha)-positive MCF-7 cells and ER-alpha-negative MDA-MB-231 human breast cancer cells. A contribution of ER to the proliferative effect of DHT in MCF-7 cells was supported by actions of small interfering RNA (siRNA) ER-alpha transfection and of the specific inhibitor of ER, ICI 182,780 to block DHT-induced proliferation. A contribution of the possible conversion of DHT to androstane-3alpha, 17beta-diol was not excluded in these MCF-7 cell studies. In MDA-MB-231 cells, a novel mechanism was implicated, in that anti-integrin alphavbeta3 or an Arg-Gly-Asp (RGD) peptide targeted at a small molecule binding domain of the integrin eliminated the DHT effect on cell proliferation. Anti-integrin alphavbeta3 did not affect DHT action on MCF-7 cells. A contribution from classical androgen receptor to the DHT effect in each cell line was excluded. A proliferative DHT signal is transduced in both ER-alpha-positive and ER-alpha-negative breast cancer cells, but by discrete mechanisms.

Modification of survival pathway gene expression in human breast cancer cells by tetraiodothyroacetic acid (tetrac).

Tetraiodothyroacetic acid (tetrac) inhibits the cellular actions of thyroid hormone initiated at the hormone receptor on plasma membrane integrin alphavbeta3. Via interaction with the integrin, tetrac is also capable of inhibiting the angiogenic effects of vascular endothelial growth factor and basic fibroblast growth factor. MDA-MB-231 cells are estrogen receptor-negative human breast cancer cells shown to be responsive to tetrac in terms of decreased cell proliferation. Here we describe actions initiated at the cell surface receptor by unmodified tetrac and nanoparticulate tetrac on a panel of survival pathway genes in estrogen receptor-negative human breast cancer (MDA-MB-231) cells. Nanoparticulate tetrac is excluded from the cell interior. Expression of apoptosis inhibitors XIAP (X-linked inhibitor of apoptosis) and MCL1 (myeloid cell leukemia sequence 1) was downregulated by nanoparticulate tetrac in these breast cancer cells whereas apoptosis-promoting CASP2 and BCL2L14 were upregulated by the nanoparticulate formulation. Unmodified tetrac affected only XIAP expression. Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene was increased by both formulations of tetrac, as was the expression of CBY1, a nuclear inhibitor of catenin activity. The majority of differentially regulated Ras-oncogene family members were downregulated by nanoparticulate tetrac. The latter downregulated expression of epidermal growth factor receptor gene and unmodified tetrac did not. Nanoparticulate tetrac has coherent anti-cancer actions on expression of differentially-regulated genes important to survival of MDA-MB-231 cells.

L-Thyroxine vs. 3,5,3'-triiodo-L-thyronine and cell proliferation: activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase.

3,5,3'-Triiodo-l-thyronine (T(3)), but not l-thyroxine (T(4)), activated Src kinase and, downstream, phosphatidylinositol 3-kinase (PI3-kinase) by means of an alpha(v)beta(3) integrin receptor on human glioblastoma U-87 MG cells. Although both T(3) and T(4) stimulated extracellular signal-regulated kinase (ERK) 1/2, activated ERK1/2 did not contribute to T(3)-induced Src kinase or PI3-kinase activation, and an inhibitor of PI3-kinase, LY-294002, did not block activation of ERK1/2 by physiological concentrations of T(3) and T(4). Thus the PI3-kinase, Src kinase, and ERK1/2 signaling cascades are parallel pathways in T(3)-treated U-87 MG cells. T(3) and T(4) both caused proliferation of U-87 MG cells; these effects were blocked by the ERK1/2 inhibitor PD-98059 but not by LY-294002. Small-interfering RNA knockdown of PI3-kinase confirmed that PI3-kinase was not involved in the proliferative action of T(3) on U-87 MG cells. PI3-kinase-dependent actions of T(3) in these cells included shuttling of nuclear thyroid hormone receptor-alpha (TRalpha) from cytoplasm to nucleus and accumulation of hypoxia-inducible factor (HIF)-1alpha mRNA; LY-294002 inhibited these actions. Results of studies involving alpha(v)beta(3) receptor antagonists tetraiodothyroacetic acid (tetrac) and Arg-Gly-Asp (RGD) peptide, together with mathematical modeling of the kinetics of displacement of radiolabeled T(3) from the integrin by unlabeled T(3) and by unlabeled T(4), are consistent with the presence of two iodothyronine receptor domains on the integrin. A model proposes that one site binds T(3) exclusively, activates PI3-kinase via Src kinase, and stimulates TRalpha trafficking and HIF-1alpha gene expression. Tetrac and RGD peptide both inhibit T(3) action at this site. The second site binds T(4) and T(3), and, via this receptor, the iodothyronines stimulate ERK1/2-dependent tumor cell proliferation. T(3) action here is inhibited by tetrac alone, but the effect of T(4) is blocked by both tetrac and the RGD peptide.

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant.

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G(2) checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-Delta (null) mutant is defective in both S and G(2) checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G(2) checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1 and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G(2) checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G(2)-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G(2) arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.

Activation of the budding yeast securin Pds1 but not Rad53 correlates with double-strand break-associated G2/M cell cycle arrest in a mec1 hypomorphic mutant.

Budding yeast Mec1, encoded by the yeast ATR/ATM homolog, negatively regulates cell cycle progression by activating Rad53 (Chk2) and Chk1, two parallel downstream checkpoint pathways. Chk1 phosphorylates Pds1 (securin), which prevents Pds1 degradation. We determined whether activation of both downstream pathways is required to establish G2 arrest in response to double-strand breaks (DSBs). In a hypomorphic mec1 mutant, Rad53 activation was not required to establish G2 arrest triggered by a single HO endonuclease-generated DSB. However, Pds1 phosphorylation did correlate with G2 arrest and mec1-21 pds1 cells did not arrest in G2 after exposure to ionizing radiation. The G2 checkpoint genes, CHK1 and PDS1, did confer radiation resistance in mec1-21, indicating that CHK1-mediated pathway is functional in the mec1 hypomorph. Thus, phosphorylation of Pds1 but not Rad53 correlates with G2 arrest in response to DSBs in the mec1 hypomorphic mutant.

Reduced expression of p27kip1 and increased hepatocyte proliferation in p53-deficient mice.

Livers from wild-type and p53-deficient mice were analyzed for the expression of cell-cycle regulatory proteins in an attempt to determine the mechanism for the increased proliferation of liver cells in p53-deficient mice associated with enhanced susceptibility to aflatoxin-induced liver cancer. The most striking difference found was a significant reduction of the cyclin-dependent kinase inhibitor p27(kip1) in the livers of 3-mo-old p53-/- mice, whereas only small changes were found in the expression of cyclins, cyclin-dependent kinases, and the inhibitors p21(cip1) and p16(ink4a). Relative to wild-type liver, the amounts of p27(kip1) mRNA were reduced at both 1 and 3 mo, whereas the levels of p27(kip1) protein were decreased only at 3 mo. These results identify an uncharacterized link between the expression of p53 and p27(kip1) that may involve both transcriptional and post-transcriptional regulation and allow hepatocytes to continue to proliferate after 3 wk of age. We postulate that this increased proliferation leads to increased susceptibility to aflatoxin-induced hepatocarcinogenesis.

Derivation, characterization, and phenotypic variation of hepatic progenitor cell lines isolated from adult rats.

Liver progenitor cells (LPCs) cloned from adult rat livers following allyl alcohol injury express hematopoietic stem cell and early hepatic lineage markers when cultured on feeder layers; under these conditions, neither mature hepatocyte nor bile duct, Ito, stellate, Kupffer cell, or macrophage markers are detected. These phenotypes have remained stable without aneuploidy or morphological transformation after more than 100 population doublings. When cultured without feeder layers, the early lineage markers disappear, and mature hepatocyte markers are expressed; mature hepatocytic differentiation and cell size are also augmented by polypeptide and steroidal growth factors. In contrast to hepatocytic potential, duct-like structures and biliary epithelial markers are expressed on Matrigel. Because they were derived without carcinogens or mutagens, these bipotential LPC lines provide novel tools for models of cellular plasticity and hepatocarcinogenesis, as well as lines for use in cellular transplantation, gene therapy, and bioreactor construction.