The role of workplace absence as an organizational mechanism in predicting employee alcohol and drug disorders

Background. EAP programs for airline pilots in companies with a well developed recovery management program are known to reduce pilot absenteeism following treatment. Given the costs and safety consequences to society, it is important to identify pilots who may be experiencing an AOD disorder to get them into treatment. ^ Hypotheses. This study investigated the predictive power of workplace absenteeism in identifying alcohol or drug disorders (AOD). The first hypothesis was that higher absenteeism in a 12-month period is associated with higher risk that an employee is experiencing AOD. The second hypothesis was that AOD treatment would reduce subsequent absence rates and the costs of replacing pilots on missed flights. ^ Methods. A case control design using eight years (time period) of monthly archival absence data (53,000 pay records) was conducted with a sample of (N = 76) employees having an AOD diagnosis (cases) matched 1:4 with (N = 304) non-diagnosed employees (controls) of the same profession and company (male commercial airline pilots). Cases and controls were matched on the variables age, rank and date of hire. Absence rate was defined as sick time hours used over the sum of the minimum guarantee pay hours annualized using the months the pilot worked for the year. Conditional logistic regression was used to determine if absence predicts employees experiencing an AOD disorder, starting 3 years prior to the cases receiving the AOD diagnosis. A repeated measures ANOVA, t tests and rate ratios (with 95% confidence intervals) were conducted to determine differences between cases and controls in absence usage for 3 years pre and 5 years post treatment. Mean replacement costs were calculated for sick leave usage 3 years pre and 5 years post treatment to estimate the cost of sick leave from the perspective of the company. ^ Results. Sick leave, as measured by absence rate, predicted the risk of being diagnosed with an AOD disorder (OR 1.10, 95% CI = 1.06, 1.15) during the 12 months prior to receiving the diagnosis. Mean absence rates for diagnosed employees increased over the three years before treatment, particularly in the year before treatment, whereas the controls’ did not (three years, x = 6.80 vs. 5.52; two years, x = 7.81 vs. 6.30, and one year, x = 11.00cases vs. 5.51controls. In the first year post treatment compared to the year prior to treatment, rate ratios indicated a significant (60%) post treatment reduction in absence rates (OR = 0.40, CI = 0.28, 0.57). Absence rates for cases remained lower than controls for the first three years after completion of treatment. Upon discharge from the FAA and company’s three year AOD monitoring program, case’s absence rates increased slightly during the fourth year (controls, x = 0.09, SD = 0.14, cases, x = 0.12, SD = 0.21). However, the following year, their mean absence rates were again below those of the controls (controls, x = 0.08, SD = 0.12, cases, x¯ = 0.06, SD = 0.07). Significant reductions in costs associated with replacing pilots calling in sick, were found to be 60% less, between the year of diagnosis for the cases and the first year after returning to work. A reduction in replacement costs continued over the next two years for the treated employees. ^ Conclusions. This research demonstrates the potential for workplace absences as an active organizational surveillance mechanism to assist managers and supervisors in identifying employees who may be experiencing or at risk of experiencing an alcohol/drug disorder. Currently, many workplaces use only performance problems and ignore the employee’s absence record. A referral to an EAP or alcohol/drug evaluation based on the employee’s absence/sick leave record as incorporated into company policy can provide another useful indicator that may also carry less stigma, thus reducing barriers to seeking help. This research also confirms two conclusions heretofore based only on cross-sectional studies: (1) higher absence rates are associated with employees experiencing an AOD disorder; (2) treatment is associated with lower costs for replacing absent pilots. Due to the uniqueness of the employee population studied (commercial airline pilots) and the organizational documentation of absence, the generalizability of this study to other professions and occupations should be considered limited. ^ Transition to Practice. The odds ratios for the relationship between absence rates and an AOD diagnosis are precise; the OR for year of diagnosis indicates the likelihood of being diagnosed increases 10% for every hour change in sick leave taken. In practice, however, a pilot uses approximately 20 hours of sick leave for one trip, because the replacement will have to be paid the guaranteed minimum of 20 hour. Thus, the rate based on hourly changes is precise but not practical. ^ To provide the organization with practical recommendations the yearly mean absence rates were used. A pilot flies on average, 90 hours a month, 1080 annually. Cases used almost twice the mean rate of sick time the year prior to diagnosis (T-1) compared to controls (cases, x = .11, controls, x = .06). Cases are expected to use on average 119 hours annually (total annual hours*mean annual absence rate), while controls will use 60 hours. The cases’ 60 hours could translate to 3 trips of 20 hours each. Management could use a standard of 80 hours or more of sick time claimed in a year as the threshold for unacceptable absence, a 25% increase over the controls (a cost to the company of approximately of $4000). At the 80-hour mark, the Chief Pilot would be able to call the pilot in for a routine check as to the nature of the pilot’s excessive absence. This management action would be based on a company standard, rather than a behavioral or performance issue. Using absence data in this fashion would make it an active surveillance mechanism. ^

ROLE OF GSH METABOLISM IN MEDIATING STROMAL-LEUKEMIA INTERACTION AND PROMOTING CELL SURVIVAL AND DRUG RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. My dissertation works proved a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells promoted GSH synthesis in CLL cells through a novel biochemical mechanism. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. I showed that CLL cells had limited ability to take up extracellular cystine for GSH synthesis due to their low expression of the transporter Xc-, but had normal ability to uptake cysteine. In the co-culture system, the bone marrow stromal cells effectively took up cystine and reduced it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells significantly protected the leukemia cells from stress-induced apoptosis, and rendered them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach potently sensitized CLL cells to drug treatment, and effectively enhanced the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. This study reveals a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance in vivo.

The nonsteroidal anti-inflammatory drug indomethacin induces heterogeneity in lipid membranes: potential implication for its diverse biological action.

BACKGROUND: The nonsteroidal anti-inflammatory drug (NSAID), indomethacin (Indo), has a large number of divergent biological effects, the molecular mechanism(s) for which have yet to be fully elucidated. Interestingly, Indo is highly amphiphilic and associates strongly with lipid membranes, which influence localization, structure and function of membrane-associating proteins and actively regulate cell signaling events. Thus, it is possible that Indo regulates diverse cell functions by altering micro-environments within the membrane. Here we explored the effect of Indo on the nature of the segregated domains in a mixed model membrane composed of dipalmitoyl phosphatidyl-choline (di16:0 PC, or DPPC) and dioleoyl phosphatidyl-choline (di18:1 PC or DOPC) and cholesterol that mimics biomembranes. METHODOLOGY/PRINCIPAL FINDINGS: Using a series of fluorescent probes in a fluorescence resonance energy transfer (FRET) study, we found that Indo induced separation between gel domains and fluid domains in the mixed model membrane, possibly by enhancing the formation of gel-phase domains. This effect originated from the ability of Indo to specifically target the ordered domains in the mixed membrane. These findings were further confirmed by measuring the ability of Indo to affect the fluidity-dependent fluorescence quenching and the level of detergent resistance of membranes. CONCLUSION/SIGNIFICANCE: Because the tested lipids are the main lipid constituents in cell membranes, the observed formation of gel phase domains induced by Indo potentially occurs in biomembranes. This marked Indo-induced change in phase behavior potentially alters membrane protein functions, which contribute to the wide variety of biological activities of Indo and other NSAIDs.

Studies of the mechanism of daunorubicin carcinogenesis: Modulation by vitamin E

Daunorubicin (DNR) is an anthracycline antibiotic used as a cancer chemotherapeutic agent. However, it causes mammary adenocarcinomas in female Sprague-Dawley (SD) rats. Vitamin E (E) has been found to reduce DNR carcinogenicity. I investigated the mechanism of DNR carcinogenicity and its interaction with E in SD rats by studying DNR-DNA adduct formation and the influence of E status on DNR clearance and free radical producing and detoxifying enzymes.^ The hypothesis was that DNR exerts its tumorigenic effect via free radicals generated during redox cycling and production of reactive intermediates capable of forming DNA adducts. E was postulated to act as a protective agent through a combination of its antioxidant property, modulation of drug clearance and levels of free radical producing and detoxifying enzymes.^ DNA adduct formation was measured by the nuclease P1 $\sp{32}$P-post labeling assay. In vitro, DNR was activated by rat liver microsomes and either NADPH or cumene hydrogen peroxide (CuOOH). Rat liver DNA incubated with this mixture formed two adducts when the cofactor was NADPH and three adducts when CuOOH was used. In vivo, SD rats were treated with i.v. doses of DNR. No detectable DNR-DNA adducts were formed in liver or mammary DNA in vivo, although there was an intensification of endogenous DNA adducts.^ Groups, 1, 2, 3 and 4 of weanling female SD rats were fed 0, 100, 1,000 and 10,000 mg $\alpha$-tocopheryl acetate/kg diet respectively. A comparison of Groups 1 and 4 showed no effect of E status on clearance of 10 mg tritiated DNR/kg body weight over 72 hours. However, liver cleared DNR at a faster rate than mammary epithelial cells (MEC).^ Xanthine oxidase, which catalyzes DNR redox cycling, was significantly decreased in liver and MEC of rats in group 4 compared to groups 1, 2, and 3. Detoxifying enzymes were not dramatically affected by E supplementation. Quinone reductase in MEC was significantly increased in group 4 compared to other groups. Overall, the liver had higher levels of free radical detoxifying enzymes compared to MEC.^ These data support a role of free radicals in DNR carcinogenicity because (1) endogenous DNA adducts formed due to free radical insult are further intensified by DNR treatment in vivo, (2) MEC, the specific target of DNR carcinogenicity, cannot rapidly clear DNR and have a lower free radical detoxifying capability than liver, (3) E supplementation caused lowering of free radical generating potential via xanthine oxidase, and increased DNR detoxification due to elevation of quinone reductase in MEC. ^

The biology and mechanism of chemoresistance of brain metastases

Primary brain neoplasms and metastases to the brain are generally resistant to systemic chemotherapy. The purpose of theses studies was to determine the mechanism(s) for this resistance. We have developed a model to study the biology of brain metastasis by injecting metastatic K1735 melanoma cells into the carotid artery of syngeneic C3H/HeN or nude mice. The resulting brain lesions are produced in the parenchyma of the brain. Mice with subcutaneous or brain melanoma lesions were treated intravenously with doxorubicin (DXR) (7 mg/kg). The s.c. lesions regressed in most of the mice whereas no therapeutic benefits were produced in mice with brain metastases. The intravenous injection of sodium fluorescine revealed that the blood-brain barrier (BBB) is intact in and around brain metastases smaller than 0.2 mm$\sp2$ but not in larger lesions, implying that the BBB is not a major obstacle for chemotherapy of brain metastases.^ Western blot and FACS analyses revealed that K1735 melanoma brain metastases expressed high levels of P-glycoprotein (P-gp) as compared to s.c. tumors or in vitro cultures. Similarly, K1735 cells from brain metastases expressed higher levels of mdrl mRNA. This increased expression of mdrl was due to adaptation to the local brain environment. We base this conclusion on the results of two studies. First, K1735 cells from brain metastases cultured for 7 days lost the high mdrl expression. Second, in crossover experiments K1735 cells from s.c. tumors (low mdrl expression) implanted into the brain exhibited high levels of mdrl expression whereas cells from brain metastases implanted s.c. lost the high level mdrl expression.^ To investigate the mechanism by which the brain environment upregulates mdrl expression of the K1735 cells we first studied the regulation of P-gp in brain endothelial cells. Since astrocytes are closely linked with the BBB we cocultured brain endothelial cells for 3 days with astrocytes. These endothelial cells expressed high levels of mdrl mRNA and protein whereas endothelial cells cocultured with endothelial cells or fibroblasts did not. We next cocultured K1735 melanoma cells with astrocytes. Here again, astrocytes (but not fibroblasts or tumor cells) uprelated the mdrl expression in K1735 tumor cells. This upregulation inversely correlated with intracellular drug accumulation and sensitivity to DXR.^ The data conclude that the resistance of melanoma brain metastases to chemotherapy is not due to an intact BBB but to the upregulation of the mdrl gene by the organ microenvironment, i.e., the astrocytes. This epigenetic mediated resistance to chemotherapy has wide implications for the therapy of brain metastases. ^

Modulating bioreductive drug activity with antivascular agents

Modulation of tumor hypoxia to increase bioreductive drug antitumor activity was investigated. The antivascular agent 5,6-dimethylxanthenone acetic acid (DMXAA) was used in combination studies with the bioreductive drugs Tirapazamine (TPZ) and Mitomycin C (MMC). Blood perfusion studies with DMXAA showed a maximal reduction of 66% in tumor blood flow 4 hours post drug administration. This tumor specific decrease in perfusion was also found to be dose-dependent, with 25 and 30 mg/kg DMXAA yielding greater than 50% reduction in tumor blood flow. Increases in antitumor activity with combination therapy (bioreductive drugs $+$ DMXAA) were significant over individual therapies, suggesting an increased activity due to increased hypoxia induced by DMXAA. Combination studies yielded the following significant tumor growth delays over control: MMC (5mg/kg) $+$ DMXAA (25mg/kg) = 20 days, MMC (2.5mg/kg) $+$ DMXAA (25 mg/kg) = 8 days, TPZ (21.4mg/kg) $+$ DMXAA (17.5mg/kg) = 4 days. The mechanism of interaction of these drugs was investigated by measuring metabolite production and DNA damage. 'Real time' microdialysis studies indicated maximal metabolite production at 20-30 minutes post injection for individual and combination therapies. DNA double strand breaks induced by TPZ $\pm$ DMXAA (20 minutes post injection) were analyzed by pulsed field gel electrophoresis (PFGE). Southern blot analyses and quantification showed TPZ induced DNA double strand breaks, but this effect was not evident in combination studies with DMXAA. Based on these data, combination studies of TPZ $+$ DMXAA showed increased antitumor activity over individual drug therapies. The mechanism of this increased activity, however, does not appear to be due to an increase in TPZ bioreduction at this time point. ^

The mechanism of the pharmacological actions of dexrazoxane on different tissue types

Approximately 6,600 people die from acute myelogenous leukemia (AML) on an annual basis. During the past 10 to 15 years, there has been gradual overall improvements in the therapy of this disease, yet the majority of patients with AML succumb to this disease. In an attempt to improve current therapeutic strategies for AML, we became interested in a commercially available drug, dexrazoxane, which protects against anthracycline-induced cardiotoxicity. We have investigated dexrazoxane's (DEX) effects on different tissue types in an effort to determine its unique mechanism of action. Colony forming assays were used to evaluate stem-cell renewal of myeloid cells in vitro and median effect analysis was used to evaluate antagonism, synergism, or additivity. The anthracyclines, doxorubicin, daunorubicin, and idarubicin were individually combined with DEX in leukemic myeloid models to determine if the combination of the two drugs resulted in a synergistic, additive or antagonistic effect. Etoposide and cytosine arabinoside were also evaluated in combination with DEX using the same in vitro model and evaluated similarly. ^ Dexrazoxane in combination with any of the anthracyclines was schedule dependent. The combination of DEX and anthracycline resulted in a greater antitumor effect than anthracycline alone except for DEX administered 24 hours before doxorubicin or daunorubicin. These data were corroborated through median effect analysis. Etoposide in combination with dexrazoxane was synergistic for all combinations, and the combination of cytosine arabinoside and DEX was schedule dependent. In contrast, using an in vivo gastrointestinal model, DEX in combination with doxorubicin was antagonistic for almost all of the ratios used, except for the highest. A Withers' assay was used to evaluate toxicity on jejunal crypt cells. No effect was apparent for the combination of idarubicin and DEX, however, as seen with RZ, DEX in addition to radiation greatly potentiated the cytotoxic effects of radiation on crypts. These paradoxical effects of dexrazoxane were initially enigmatic, but after additional investigation, we propose a model that explains our findings. We conclude that DEX in combination with anthracyclines produces an additive to synergistic antileukemic response and may have therapeutic potential clinically. Additionally, DEX protects the gastrointestinal tract from doxorubicin toxicity, which could have clinical implications for the administration of greater doses of doxorubicin. ^

The relationship of mutations in alpha- and beta-tubulin to microtubule assembly and anti-mitotic drug resistance

The mechanisms responsible for anti-cancer drug (including Taxol) treatment failure have not been identified. In cell culture model systems, many β-tubulin, but very few α-tubulin, mutations have been associated with resistance to Taxol. To test what, if any, mutations in α-tubulin can cause resistance, we transfected a randomly mutagenized α-tubulin cDNA into Chinese hamster ovary (CHO) cells and isolated drug resistant cell lines. A total of 12 mutations were identified in this way and all of them were confirmed to confer Taxol resistance. Furthermore, all cells expressing mutant α-tubulin had less microtubule polymer. Some cells also had abnormal nuclei and enlarged cell bodies. The data indicate that α-tubulin mutations confer Taxol resistance by disrupting microtubule assembly, a mechanism consistent with a large number of previously described β-tubulin mutations. ^ Because α- and β-tubulin are almost identical in their three dimensional structure, we hypothesized that mutations discovered in one subunit, when introduced into the other, would produce similar effects on microtubule assembly and drug resistance. 9 α- and 2 β-tubulin mutations were tested. The results were complex. Some mutations produced similar changes in microtubule assembly and drug resistance irrespective of the subunit in which they were introduced, but others produced opposite effects. Still one mutation produced resistance when present in one subunit, yet had no effect when present on the other; and one mutation that produced Taxol resistance when present in α-tubulin, resulted in assembly-defective tubulin when it was present in β-tubulin. The results suggest that in most cases, the same amino acid modification in α- and β-tubulin affects the microtubule structure and assembly in a similar way. ^ Finally, we tested whether three β-tubulin mutations found in patient tumors could confer resistance to Taxol by recreating the mutations in a β-tubulin cDNA and transfecting it into CHO cells. We found that all three mutations conferred Taxol resistance, but to different extents. Again, microtubule assembly in the transfectants was disrupted, suggesting that mutations in β-tubulin are a potential problem in cancer therapeutics. ^

A novel mechanism of 3-bromopyruvate-induced cell death through targeting hexokinase and ubiquitination

Increased dependence on aerobic glycolysis for energy (ATP) supply has been observed in various human cancer cells. It is plausible to exploit this metabolic alteration for therapeutic benefits by inhibiting glycolysis to preferentially abolish cancer energy metabolism and kill the malignant cells. 3-Bromopyruvate has been shown to be a potent inhibitor of glycolysis capable of inducing severe ATP reduction and cell death in various cancer cell lines, especially cancer cells with mitochondrial defects or under hypoxic conditions. However, the detailed mechanisms of this novel anticancer agent still remain unclear. My study demonstrated that 3-Bromopyruvate caused a covalent modification of hexokinase II, a key glycolytic enzyme, and disrupted its association with mitochondria. This led to mitochondrial permeability transition and a substantial release of apoptosis-inducing faction (AIF) prior to cytochrome c release. Dissociation of HK II from mitochondria using a cell permeable specific peptide also induced the release of AIF and cytochrome c, and caused substantial cell death. HK II-targeted peptide did not cause significant change in mitochondria respiration and glycolysis activity, suggesting that dissociation of this molecule from mitochondria alone can also cause cell death, and that this may be a novel mechanism by which 3-Bromopyruvate exerts its potent cytotoxic action, in addition to its inhibition of the enzyme activity. Another significant new discovery was that 3-Bromopyruvate induced rapid reduction of protein ubiquitination in vivo, which occurred within several hours of drug incubation and before ATP reduction and cell death. Further mechanistic studies showed that this was due to the inhibition the ubiquitin activating enzyme E1 and the conjugating enzyme E2. Knocking down ubiquitin protein expression by siRNA did not suppress mitochondria respiration and glycolysis, but caused significant cell death. Taken together, this study demonstrated that induction of HK II dissociation from mitochondria and inhibition of glycolysis are two newly discovered mechanisms that contribute to the potent anticancer activity of 3-Bromopyruvate, and identified this compound as a valuable chemical tool for research in protein ubiquitination. ^

Anticancer activity of OSW-1: Induction of apoptotic pathway in leukemia and autophagic death in pancreatic cancer through a calcium mediated mechanism

OSW-1 is a natural compound found in the bulbs of Orninithogalum saudersiae, a member of the lily family. This compound exhibits potent antitumor activity in vitro with the IC50 values in the low nanomolar concentration range and demonstrating its ability to kill drug resistant cancer cells. In an effort to discover the unknown mechanism of action of this novel compound as a potential anticancer agent, the main objective of this research project was to test the cytotoxicity of OSW-1 against various cancer lines, and to elucidate the biochemical and molecular mechanism(s) responsible for the anticancer activity of OSW-1. My initial investigation revealed that OSW-1 is effective in killing various cancer cells including pancreatic cancer cells and primary leukemia cells resistant to standard chemotherapeutic agents, and that non-malignant cells were less sensitive to this compound. Further studies revealed that in leukemia cells, OSW-1 causes a significant increase in cytosolic calcium and activates rapid calcium-dependent apoptosis by the intrinsic pathway. Additionally, OSW-1 treatment leads to the degradation of the ER chaperone GRP78/BiP implicated in the survival of cancer cells. Meanwhile, it shows a reduced sensitivity in respiration-deficient sub-clones of leukemia cells which had higher basal levels of Ca2+. Mechanistically, it was further demonstrated that cytosolic Ca2+ elevations were observed together with Na+ decreases in the cytosol, suggesting OSW-1 caused the calcium overload through inhibition of the Na+/Ca 2+exchanger (NCX). Although similar calcium disturbances were observed in pancreatic cancer cells, mechanistic studies revealed that autophagy served as an initial pro-survival mechanism subsequent to OSW-1 treatment but extended autophagy caused inevitable cell death. Furthermore, combination of OSW-1 with autophagy inhibitors significantly enhances the cytotoxicity against pancreatic cancer cells. Taken together, this study revealed the novel mechanism of OSW-1 which is through inhibition of the Na+/Ca2+ exchanger and provides a basis for using this compound in combination with other agents for the treatment of pancreatic cancer which is resistant to available anticancer drugs. ^

Mechanism of methylation gene reactivation in human cancer cells

Epigenetic silencing of tumor suppressor genes by DNA hypermethylation at promoter regions is a common event in carcinogenesis and tumor progression. Abrogation of methylation and reversal of epigenetic silencing is a very potent way in cancer treatment. However, the reactivation mechanisms are poorly understood. In this study, we first developed a cell line model system named YB5, derived from SW48 cancer cell line, which bears one copy of stably integrated EGFP gene on Chromosome 1p31.1 region. The GFP gene expression is transcriptionally silenced due to the hypermethylated promoter CMV. However, the GFP expression can be restored using demethylating agent 5-aza-2' deoxycytidine (DAC), and detected by FACS and fluorescent microscopy. Using this system, we observed the heterogeneous reactivation induced by DAC treatment. After flow sorting, GFP negative cells exhibited similar level of incomplete demethylation compared to GFP positive cells on repetitive LINE1 element, tumor suppressor genes such as P16, CDH13, and RASSF1a, and CMV promoter as well. However, the local chromatin of CMV-GFP locus altered to an open structure marked by high H3 lysine 9 acetylation and low H3 lysine 27 tri-methylation in GFP positive cells, while the GFP negative cells retained mostly the original repressive marks. Thus, we concluded that DAC induced DNA hypomethylation alone does not directly determine the level of re-expression, and the resetting of the local chromatin structure under hypomethylation environment is required for gene reactivation. Besides, a lentivirus vector-based shRNA screening was performed using the YB5 system. Although it is the rare chance that vector lands in the neighboring region of GFP, we found that the exogenous vector DNA inserted into the upstream region of GFP gene locus led to the promoter demethylation and reactivated the silenced GFP gene. Thus, epigenetic state can be affected by changing of the adjacent nucleic acid sequences. Further, this hypermethylation silenced system was utilized for epigenetic drug screening. We have found that DAC combined with carboplatin would enhance the GFP% yield and increase expression of other tumor suppressor genes than DAC alone, and this synergistic effect may be related to DNA repair process. In summary, these studies reveal that reversing of methylation silencing requires coordinated alterations of DNA methylation, chromatin structure, and local microenvironment. ^