Impact of Donor Specific HLA Antibody Monitoring After Kidney Transplantation.

Kidney transplantation (KT) recipients with donor specific HLA antibodies (DSA) encounter higher rates of acute rejection and inferior allograft survival. We report our single center experience with prospective DSA monitoring and provide details of treatments utilized to overcome the potential impact of DSA in a cohort of predominantly African American adult KT recipients. Seventy-five flow crossmatch negative KT recipients underwent periodic screening for DSA utilizing the single antigen bead assay at 3, 6, 9, and 12 months post-transplant. Allograft biopsies were performed in the presence of DSA and/or evidence of graft dysfunction. The incidence of DSA was 23%, with a predominance of Class II antibodies. The rate of rejection was 6 times higher in DSA positive KT recipients compared to DSA negative patients (41% versus 7%, p = 0.004). In the DSA positive group, rejections occurred exclusively in the presence of de novo DSA and were predominantly antibody-mediated or mixed rejections. Despite a higher incidence of rejection in KT recipients with DSA, there were no significant differences in serum creatinine, graft survival, and patient survival between DSA positive and negative recipients at median follow-up of 18 months. DSA positive patients had significantly higher proteinuria compared to DSA negative recipients at 6 months, 1 year, and 3 years of follow-up. In conclusion, the detrimental effects of DSA on allograft function could be mitigated by serial DSA surveillance, protocol biopsies, and alterations in immunosuppression. With these measures, the improvement in graft survival in DSA positive KT recipients, at least at short-term, is encouraging.

Mycophenolate mofetil inhibits hepatitis C virus replication in human hepatic cells.

Hepatitis C virus (HCV) infection is the most common indication for liver transplantation and the major cause of graft failure. A widely used immunosuppressant, cyclosporine A (CsA), for people who receive organ transplantation, has been recognized to have the ability to inhibit HCV replication both in vivo and in vitro. In this study, we investigated the effects of several other immunosuppressants, including mycophenolate mofetil (MMF), rapamycin and FK506, on HCV replication in human hepatic cells. MMF treatment of hepatic cells before or during HCV infection significantly suppressed full cycle viral replication, as evidenced by decreased expression of HCV RNA, protein and production of infectious virus. In contrast, rapamycin and FK506 had little effect on HCV replication. Investigation of the mechanism(s) disclosed that the inhibition of HCV replication by MMF was mainly due to its depletion of guanosine, a purine nucleoside crucial for synthesis of guanosine triphosphate, which is required for HCV RNA replication. The supplement of exogenous guanosine could reverse most of anti-HCV effect of mycophenolate mofetil. These data indicate that MMF, through the depletion of guanosine, inhibits full cycle HCV JFH-1 replication in human hepatic cells. It is of interest to further determine whether MMF is indeed beneficial for HCV-infected transplant recipients in future clinical studies.

The CCAAT box binding transcription factor, nuclear factor-Y (NF-Y) regulates transcription of human aldo-keto reductase 1C1 (AKR1C1) gene.

Dihydrodiol dehydrogenases are a family of aldo-keto reductases (AKR1Cs) involved in the metabolism of steroid hormones and xenobiotics. Herein, we have cloned and characterized the proximal promoter region of the human AKR1C1 gene. The 5' flanking proximal promoter region of the AKR1C1 gene consists of a TATA box and an inverted CCAAT binding site. Deletion analysis of the 5' flanking, approximately 3.0 kb region of the human AKR1C1 gene identified the region between -128 and -88 as the minimal proximal promoter essential for basal transcription of AKR1C1 in human ovarian (2008 and 2008/C13*), lung (H23 and A549) and liver carcinoma (HepG2) cells. Site-directed mutagenesis studies indicated that the transcription factor binding sites for NF-Y/CEBP were involved in controlling the basal transcription of AKR1C1 in all the cancer cells studied. Electrophoretic mobility shift (EMSAs) and gel-supershift assays demonstrated that the transcription factor NF-Y preferentially binds to the inverted CCAAT box at (-109)ATTGG(-105) of the AKR1C1 gene. Chromatin immunoprecipitation (ChIP) analysis confirmed the in vivo association between NF-Y and human AKR1C1 gene promoter in human ovarian, lung and liver carcinoma cells. Ectopic expression of NF-Ys increased the AKR1C1 gene transcription, whereas expression of a dominant-negative NF-YA or suppression of NF-YA decreased the AKR1C1 gene transcription. A 2-fold increase in AKR1C1 transcription was observed specifically in cisplatin-treated 2008 cells that were CCAAT box-dependent. These results indicate that the NF-Y regulates the basal transcription of AKR1C1 in human ovarian, lung and liver carcinoma cells and the cisplatin-induced transcription in human ovarian carcinoma cells.

Dihydrodiol dehydrogenases regulate the generation of reactive oxygen species and the development of cisplatin resistance in human ovarian carcinoma cells.

We have previously demonstrated that overexpression of dihydrodiol dehydrogenase isoform 1 (DDH1) or DDH2 leads to the induction of drug resistance to platinum based drugs in human ovarian, lung, cervical and germ cell tumor cell lines. DDH belongs to a family of aldoketo reductases that are involved in the detoxification of several endogenous and exogenous substrates. DDH1 and DDH2 in particular have been shown to be involved in the detoxification (activation?) of polycyclic aromatic hydrocarbons (PAH). Based on the involvement of DDH in the detoxification of electrophilic PAH intermediates, the effect of DDH on the production of reactive oxygen species (ROS) in a cisplatin-sensitive and -resistant human ovarian carcinoma cell line was investigated in the current study. In addition to the overexpression of DDH1 and DDH2, increased expression of DDH3 was demonstrated in the cisplatin-resistant 2008/C13* cells, compared to the parental 2008 cells. However, as assessed by RT-PCR, neither cell line expressed DDH4. The 2008/C13* cells were eightfold resistant to cisplatin, and transfection experiments utilizing cisplatin-sensitive 2008 cells suggest that this could be mediated by overexpression of either DDH1, DDH2, or DDH3. The 2008/C13* cells had lower basal intracellular ROS level as compared to the 2008 cells and ROS production was decreased in the recombinant 2008 cells with forced, constitutive overexpression of either, DDH1, DDH2, or DDH3. Transfection of siRNA against DDH1 or DDH2 in the cisplatin-resistant 2008/C13* cells not only significantly decreased their cisplatin-resistance index (as assayed by MTT and colony formation assay) but also led to an increase in the basal levels of ROS production (although transfection of siRNA against DDH3 resulted in cell death). The 2008/C13* cells were found to be cross-resistant to the cytotoxic effects of hydrogen peroxide and tert-butyl hydroperoxide and knockdown of either DDH1 or DDH2 expression (using siRNA) resulted in sensitization of the resistant cells to these agents. These results support the conclusion that the increased levels of DDH in the 2008/C13* cells are directly responsible for the reduced production of ROS and that this may play a role in the development of cisplatin resistance.

Differential partitioning of Galphai1 with the cellular microtubules: a possible mechanism of development of Taxol resistance in human ovarian carcinoma cells.

BACKGROUND: Taxol binds to the cellular microtubules and suppresses their dynamic instability. Development of tumor cell resistance to taxol is typically associated with increased expression of the drug efflux pump P-glycoprotein and/or alterations in the microtubules. Recently, changes in the dynamic instability of the microtubules have also been associated with development of taxol resistance in a lung cancer cell line. We have established a 250-fold taxol-resistant human ovarian carcinoma subline (2008/13/4) that does not display the typical alterations associated with development of drug resistance. RESULTS: Utilizing the mRNA differential display technique, we observed increased expression of an alpha subunit of the guanine nucleotide-binding protein, Galphai1, in the taxol-resistant human ovarian carcinoma cell lines compared to the parental 2008 cells. Several isoforms of the alpha-subunit of the G protein have been identified and the Galphai (inhibitory) are so named because they inhibit the activity of adenylate cyclase leading to inactivation of the cAMP-dependent protein kinase A (PKA) pathway. In addition, Galphai1 is also known to bind to microtubules and activates their GTPase activity and thus induces depolymerization of the microtubules. In the present study we demonstrate that the intracellular level of cAMP and the PKA activity were higher in the taxol-resistant 2008/13/4 and the 2008/17/4 cells despite the increased expression of Galphai1 in these cells. Moreover, Galphai1 was found to be localized not on the cell membrane, but in intracellular compartments in both the taxol-sensitive and -resistant human ovarian carcinoma cells. Interestingly, increased association of the Galphai1 protein and the microtubules in the taxol-resistant cells compared to the parental 2008 cells was observed, both prior to and after treatment of these cells with taxol. CONCLUSION: Based on the opposing effects of taxol and the Galphai1 protein on the microtubule dynamic instability (taxol suppresses microtubule dynamic instability whilst the Galphai1 protein inhibits the suppression) our results indicate the operation of a novel pathway that would enable the cells to escape the cytotoxic effects of taxol.

Ubiquitous induction of resistance to platinum drugs in human ovarian, cervical, germ-cell and lung carcinoma tumor cells overexpressing isoforms 1 and 2 of dihydrodiol dehydrogenase.

We have recently demonstrated that overexpression of dihydrodiol dehydrogenase (DDH) in human ovarian carcinoma cells (2008/C13*) is associated with cisplatin and carboplatin resistance. Furthermore, we have also elucidated that transfection of parental human ovarian carcinoma cells with a full-length DDH1 cDNA leads to induction of resistance to the platinum drugs. The development of cisplatin resistance in the transfected cells is associated with an increase in DDH enzyme activity. Previous studies have identified several different mechanisms for development of cisplatin resistance, including altered DNA repair capacity, increased GSH-based detoxification, and increased metallothionein content. However, none of these mechanisms has been found to be universally associated with the development of cisplatin resistance in tumor cells from different tissue sources. The present study was undertaken to assess whether overexpression of DDH1 or DDH2 (in human ovarian, cervical, lung and germ-cell tumor cell lines) could specifically induce resistance to the platinum drugs in these cell lines. We demonstrated a ubiquitous association of increased expression of DDH1 or DDH2 (as judged by increased enzyme activity in transfected clones) with development of resistance to cisplatin and carboplatin. Moreover, we also found a lack of cross-resistance to anticancer drugs that have a different mode of action including paclitaxel, vincristine, doxorubicin hydrochloride, and melphalan. Although at present it is not clear how DDH is involved in platinum drug resistance, the identification of this gene as a causal factor in a series of cell lines derived from different tumors with different intracellular compositions indicates the importance of deciphering this hitherto undefined pathway which can produce resistance to platinum drugs.

Overexpression of sorcin, a calcium-binding protein, induces a low level of paclitaxel resistance in human ovarian and breast cancer cells.

Paclitaxel, an antimitotic, anticancer agent, induces cell cycle arrest in the mitotic phase by binding to the beta-tubulin subunit and forming highly stable microtubule polymers that resist depolymerization. The overexpression of the P-glycoprotein (P-gp) and/or alteration in the cellular microtubules is associated with the development of paclitaxel resistance. However, we have established a paclitaxel-resistant human ovarian carcinoma subline (2008/13/4) wherein the degree of resistance could not be correlated with overexpression of P-gp, alterations in the alpha- and beta-tubulin isotypes, or changes in the drug-binding affinity of the microtubules. mRNA differential display analysis revealed the overexpression of sorcin, a calcium-binding protein in the 2008/13/4 cells. However, no detectable changes in the intracellular calcium levels were detected in the parental and the paclitaxel-resistant variant. Furthermore, co-treatment with A23187, a calcium ionophore, did not alter the cytotoxicity of paclitaxel against the parental and the paclitaxel-resistant cells. Transfection of the parental 2008 cells with full-length sorcin cDNA induced a low level (3-5-fold) of paclitaxel resistance. In addition, transfection of human breast cancer cells with the full-length sorcin cDNA also led to the induction of a low level of paclitaxel resistance in the transfectants. Although the overexpression of sorcin did not produce high levels of paclitaxel resistance, the results obtained present compelling evidence of the involvement of sorcin in developing low-level paclitaxel resistance in a variety of tumor cells. The precise biochemical mechanism(s) by which sorcin overexpression induces low-level paclitaxel resistance is currently under investigation.

Increased expression of dihydrodiol dehydrogenase induces resistance to cisplatin in human ovarian carcinoma cells.

We employed cDNA microarrays to identify the differentially expressed genes in a cisplatin-sensitive parental (2008) human ovarian carcinoma cell line and its cisplatin-resistant variant (2008/C13*). Differential expression of five genes was found in the 2008/C13* cells, a result confirmed by semi-quantitative reverse transcription-PCR. The five genes were identified as fibroblast muscle-type tropomyosin and skeletal muscle-type tropomyosin, dihydrodiol dehydrogenase, apolipoprotein J and glucose-6-phosphate dehydrogenase variant-A. Treatment of the 2008 cells with cisplatin (at its IC(50) concentration of 2 microm) induced expression of these genes, as determined by semi-quantitative reverse transcription-PCR analysis using gene-specific primers. In contrast, treatment of the drug-resistant 2008/C13* cells with cisplatin (at its IC(50) concentration of 20 microm) did not lead to the induction of any of the aforementioned genes. Most importantly, constitutive overexpression of dihydrodiol dehydrogenase (but not the other genes) in the 2008 cells led to induction of cisplatin resistance, clearly indicating its role in the development of the resistance phenotype in the 2008/C13* cells. The development of cisplatin resistance in the transfected cells was associated with an increase in the dihydrodiol dehydrogenase enzyme activity. Although at present it is not clear how dihydrodiol dehydrogenase is involved in cisplatin resistance, the identification of this gene as a causal factor suggests the existence of a hitherto undefined pathway resulting in cisplatin resistance.