The fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions. Specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug. Among the genetic variations that have been investigated in dogs, the multidrug resistance gene (MDR) is the best studied. However, other genes such as CYP1A2 and CYP2B11 control the protein syntheses involved in the metabolism of many drugs. In the present study, the MDR-1, CYP1A2 and CYP2B11 genes were examined to identify SNP polymorphisms associated with these genes in the following four canine breeds: Uruguayan Cimarron, Border Collie, Labrador Retriever and German Shepherd. The results revealed that several SNPs of the CYP1A2 and CYP2B11 genes are potential targets for drug sensitivity investigations.
Animals ; Aryl Hydrocarbon Hydroxylases/*genetics/metabolism ; Cytochrome P-450 CYP1A2/*genetics/metabolism ; Dogs/*genetics/metabolism ; P-Glycoprotein/*genetics/metabolism ; *Polymorphism, Single Nucleotide ; Steroid Hydroxylases/*genetics/metabolism

In pancreatic acinar cells Ca(2+)-dependent secretagogues promote the fusion of zymogen granules (ZG) with the apical plasma membrane (PM) and exocytosis of digestive enzymes. In addition to exocytotic fusion complexes between SNARE proteins in the ZG membrane (ZGM) and the apical PM, enzyme secretion elicited by Ca(2+)-dependent secretagogues requires cytosolic Cl and K+ and is inhibited by blockers of Cl- and K+-channels. We have identified a Cl-conductance activated by ATP, and a K+-conductance (with properties similar to ATP-sensitive K+-channels), regulated by the granule matrix protein Zg-16p in the ZGM. Both conductances are inversely regulated by a 65-kD mdr1 gene product. We have also identified a novel Ca(2+)-activated anion conductance in ZGM, the Ca(2+)-sensitivity of which increases 50-fold when Cl is replaced by 1. This conductance is blocked by micromolar H2-DIDS or DTT, reminiscent of a family of epithelial Ca(2+)-activated Cl -channels (CaCC). Expression of a CaCC in exocrine pancreas has been confirmed by RT-PCR analysis, and by immunoblotting and immunogold labeling of ZG membranes. These data suggest that ion channels in the ZGM are essential elements in pancreatic exocytosis.
Animal ; Chloride Channels/metabolism* ; Chloride Channels/genetics ; Exocytosis/physiology* ; Gene Expression/physiology ; P-Glycoprotein/metabolism ; P-Glycoprotein/genetics ; Pancreas/secretion* ; Pancreas/cytology ; Potassium Channels/metabolism* ; Potassium Channels/genetics ; Secretory Vesicles/secretion ; Secretory Vesicles/metabolism* ; Support, U.S. Gov't, P.H.S.

The mutation and reduction of mitochondrial DNA (mtDNA) have been suggested as factors in the carcinogenesis. However, whether the depletion of mtDNA induces multidrug resistance in cancer cells has not been fully investigated. To elucidate the association of cellular mtDNA content and drug resistance, we generated HCT-8 colon cancer cells which revealed a marked decrease in cellular mtDNA and ATP content, concomitant with a lack of mRNAs encoded by mtDNA. The mtDNA-depleted cells showed a decreased sensitivity and accumulation of anti-cancer drugs, suggesting that mtDNA depletion could develop multidrug resistance (MDR) phenotype in HCT-8 cells. We found that the expression level of MDR1 mRNA and its translated product P-glycoprotein was increased in the mtDNA- depleted cells, indicating that the decrease of sensitivity and accumulation of anti-cancer drug in the mtDNA-depleted cells might be due to a substantial increase in the expression of P-glycoprotein. Furthermore, increased expression of MDR1 mRNA and P-glycoprotein was due to an increase of mRNA stability rather than transcriptional activation. Taken together, these results indicate that mtDNA depletion can induce an increased P-glycoprotein expression via an increase of mRNA stability and suggest that the mtDNA depletion in cancer cells plays an important role in the induction of MDR phenotype.
Cell Line, Tumor ; DNA, Mitochondrial/*metabolism ; Doxorubicin/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; P-Glycoprotein/*genetics/metabolism ; Paclitaxel/pharmacology ; Promoter Regions, Genetic/genetics ; *RNA Stability/drug effects ; RNA, Messenger/genetics/metabolism ; Up-Regulation/drug effects/*genetics

MDR1 promoter has been shown to contain heat shock elements (HSE), and it has been reported that FM3A/M and P388/M MDR cells show a constitutively activated heat shock factor (HSF), suggesting that HSF might be an important target for reversing the multidrug resistance. Therefore, it was examined whether quercetin, which has been shown to interfere with the formation of the complex between HSE and HSF, and to downregulate the level of HSF1, can sensitize MDR cells against anticancer drugs by inhibition of HSF DNA-binding activity. In this study, quercetin appeared to inhibit the constitutive HSF DNA-binding activity and the sodium arsenite-induced HSF DNA-binding activity in the MDR cells. The basal and sodium arsenite-induced MDRCAT activities were remarkably suppressed by the treatment of quercetin. These results were well consistent with the finding that the treatment of quercetin decreased the expression level of P-gp, MDR1 gene product, in dose-dependent manner, and markedly increased the sensitivity of MDR cells to vincristine or vinblastine. These results suggest that quercetin can decrease the expression of P-gp via inhibition of HSF DNA-binding activity, and might be useful as a chemosensitizer in MDR cells.
Animal ; Antineoplastic Agents/pharmacology ; Arsenites/pharmacology ; Carcinoma/drug therapy ; Drug Resistance, Multiple/physiology* ; Drug Resistance, Neoplasm/physiology ; Heat-Shock Proteins/metabolism ; Heat-Shock Proteins/drug effects* ; Heat-Shock Proteins/antagonists & inhibitors ; Leukemia, Experimental/drug therapy ; Mice ; P-Glycoprotein/genetics ; P-Glycoprotein/drug effects ; Quercetin/pharmacology* ; Sodium Compounds/pharmacology ; Tumor Cells, Cultured ; Vinblastine/pharmacology ; Vincristine/pharmacology

BACKGROUND: The multidrug resistance (mdr1), multidrug resistance associated protein (mrp1), and glutathione-s-transferase (gst) pi genes have been associated with treatment failure in acute myeloid leukemia (AML). c-jun N-terminal kinase (JNK) activity is increased in response to chemotherapeutic agent. METHODS: To investigate the significance of multidrug resistance (mdr) parameters and JNK activity, bone marrow or peripheral blood cells from 52 patients with AML were analyzed. RT-PCR was performed for mdr1, mrp1, and gst pi gene expression. JNK expression and activity were measured using an immunoe- nzymatic kinase assay and a western blot method. RESULTS: High level expression of mdr1, mrp1, and gst pi mRNA was observed in 38.5%, 48.1% and 54.3% of AML cases, respectively. The remission rate was significantly low in cases with an older age (>55 yr), a high WBC count, poor chromosomal abnormalities, a high level expression of mdr1 and mrp1. The WBC count and mdr1 mRNA expression were independent predictors for the outcome to induction chemotherapy. There was a shorter duration of overall survival in the patients with an older age, a high WBC count, chromosome aberrations, high level expressions of mdr1 and mrp1 mRNA, and JNK activation. The patient's age, WBC count and chromosomal abnormalities were independent predictors for overall survivals. The majority (28/30) of AML cases did not show any levels of JNK activation except for two cases, which were associated with an extremely high WBC count, chromosomal aberration, high level expressions of mdr1, mrp1 and gst pi mRNA, and treatment resistance. CONCLUSIONS: These data indicate the influences of mdr1 and mrp1 mRNA expression on the clinical outcome of AML to induction chemotherapy. But it will be necessary to investigate further whether blast cells of AML resistant to chemotherapy retain the capacity to activate JNK, and relate to MDR parameters.
Adolescent ; Adult ; Aged ; *Drug Resistance, Multiple/genetics ; *Drug Resistance, Neoplasm/genetics ; Female ; Glutathione S-Transferase pi/genetics ; Humans ; JNK Mitogen-Activated Protein Kinases/*metabolism ; Leukemia, Myeloid, Acute/*drug therapy/genetics/metabolism ; Male ; Middle Aged ; Multidrug Resistance-Associated Proteins/genetics ; P-Glycoprotein/genetics ; RNA, Messenger/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Survival Analysis ; Treatment Outcome