Biomedical subjects

S L Gerson

Publications and source records attributed to S L Gerson.

At least 19 recordsLinked to original sources

PubMed1998

Phenotypic and functional comparison of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells.

Mesenchymal stem cells (MSCs) are a population of pluripotent cells within the bone marrow microenvironment defined by their ability to differentiate into cells of the osteogenic, chondrogenic, tendonogenic, adipogenic, and myogenic lineages. We have developed methodologies to isolate and culture-expand MSCs from human bone marrow, and in this study, we examined the MSC's role as a stromal cell precursor capable of supporting hematopoietic differentiation in vitro. We examined the morphology, phenotype, and in vitro function of cultures of MSCs and traditional marrow-derived stromal cells (MDSCs) from the same marrow sample. MSCs are morphologically distinct from MDSC cultures, and flow cytometric analyses show that MSCs are a homogeneous cell population devoid of hematopoietic cells. RT-PCR analysis of cytokine and growth factor mRNA in MSCs and MDSCs revealed a very similar pattern of mRNAs including IL-6, -7, -8, -11, -12, -14, and -15, M-CSF, Flt-3 ligand, and SCF. Steady-state levels of IL-11 and IL-12 mRNA were found to be greater in MSCs. Addition of IL-1alpha induced steady-state levels of G-CSF and GM-CSF mRNA in both cell preparations. In contrast, IL-1alpha induced IL-1alpha and LIF mRNA levels only in MSCs, further emphasizing phenotypic differences between MSCs and MDSCs. In long-term bone marrow culture (LTBMC), MSCs maintained the hematopoietic differentiation of CD34+ hematopoietic progenitor cells. Together, these data suggest that MSCs represent an important cellular component of the bone marrow microenvironment.

Bone Marrow↗

PubMed1998

Autologous CD34+ cell transplantation for patients with advanced lymphoma: effects of overnight storage on peripheral blood progenitor cell enrichment and engraftment.

In order to demonstrate the feasibility of mobilization, enrichment and engraftment of autologous peripheral blood CD34+ cells in patients with relapsed lymphoma, 59 peripheral blood progenitor cell (PBPC) collections from 21 patients were enriched for CD34+ cells using CEPRATE SC (CellPro, Bothell, WA, USA) immunoaffinity column. Following high-dose chemotherapy, a mean of 17 x 10(8) (range, 3-34) nucleated cells/kg containing 8.7 x 10(6) (0.3-26) CD34+ cells/kg were re-infused. Blood cell recovery in these patients was compared to engraftment capacity of unenriched PBPCs in a cohort of lymphoma patients treated with an identical high-dose chemotherapy regimen. Neutrophil and platelet engraftment was rapid in both groups including five patients who received < or = 1 x 10(6) CD34+ cells/kg. After infusion of CD34+ enriched cells, neutrophils exceeded 0.5 x 10(9)/l in 11 (8-14) days and platelets exceeded 20 x 10(9)/l (untransfused) in 15 (9-39) days. In order to optimize the immunoaffinity column utilization we stored the first PBPC collections overnight at 4 degrees C and combined them with the next day's collection prior to the CD34+ enrichment procedure in 11 patients. This maneuver resulted in a significant decrease in the CD34+ cell recovery (resulting in reinfusion of a mean of 42% less CD34+ cells). Although overnight storage did not affect neutrophil engraftment, platelet engraftment was prolonged in this group of patients even when > 2.0 x 10(6) CD34+ cells/kg were re-infused. The overnight storage procedure should be further evaluated for its effects on the CD34+ immunoaffinity enrichment procedure, megakaryocyte progenitors and platelet engraftment. We conclude that CD34+ cells enriched from peripheral blood result in rapid engraftment after high-dose chemotherapy in patients with advanced lymphoma that is comparable to that of patients receiving unenriched PBPCs.

Adult↗

PubMed1998

Heterogeneity of O6-alkylguanine-DNA-alkyltransferase measured by flow cytometric analysis in blood and bone marrow mononuclear cells.

Alkyltransferase (AGT) repairs alkylation at O6-guanine in DNA and is a major determinant of susceptibility to alkylating chemotherapeutic agents and carcinogens. Using a newly developed flow cytometry assay with the monoclonal anti-AGT antibody, mT3.1, we compared AGT expression in single-cell suspensions with standard biochemical and Western blot assays to validate the fluorescence-activated cell sorting (FACS) method and develop potential applications. From Chinese hamster ovary cells (CHO) transfected with human O6-methylguanine-DNA methyl-transferase cDNA, 6 CHO-O6-methylguanine-DNA methyl-transferase clones were isolated that expressed 0.3 to 64 fmol/microgram DNA (by biochemical assay) of human AGT. FACS yielded a linear relationship between mean fluorescence intensity and both AGT activity by biochemical assay and AGT protein by Western blot. Using this standard curve, FACS-analyzed AGT protein content in human peripheral blood mononuclear cells (PBMCs) from normal donors ranged from 6.1 to 12.8 fmol/microgram DNA, similar to those obtained by biochemical assay and Western blot. This suggests that the level of immunoreactive protein appears to be an accurate predictor of AGT activity in the steady state. FACS-AGT in PBMCs from normal donors had a low index of heterogeneity within the sample. In contrast, by FACS-AGT analysis of human bone marrow samples and granulocyte-colony-stimulating factor-mobilized PBMCs, AGT was lower and had an 8-fold higher index of heterogeneity than observed in PBMCs from normal donors. After treatment with O6-benzylguanine (O6-bG), Western and FACS-AGT detected significant levels of AGT protein for up to 24 h, whereas biochemical assay showed AGT activity less than 5% of the basal level. Because only the biochemical assay accurately measures net AGT activity, the AGT-FACS assay will not be useful in clinical trials to assess the efficacy of O6-bG or other AGT inhibitors. Thus, AGT-FACS can rapidly assess the heterogeneity of steady-state AGT in single-cell suspensions and may be useful for assay in lymphocytes, bone marrow cells, leukemic myeloma plasma cells, or cells transfected with the AGT gene; Western blot analysis is better for small samples such as tumor biopsies, whereas biochemical assay is best able to measure enzyme activity and its inactivation by O6-bG or other agents.

Animals↗

PubMed1997

Simultaneous determination of O6-benzylguanine and 8-oxo-O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for the quantification of O6-benzylguanine (O6BG) in human plasma was modified to include the metabolite, O6-benzyl-8-oxo-guanine (8-oxo-O6BG). O6-(p-Chlorobenzyl)guanine was used as the internal standard. Plasma samples were extracted with ethyl acetate and chromatographed on a C18 base-deactivated reversed-phase column. Separation was accomplished by gradient elution with mobile phases consisting of acetonitrile and phosphate buffer, pH 3.60. Eluted compounds were observed with diode array detection at 288 nm (O6BG) and 292 nm (8-oxo-O6BG). Standard curves were linear from 12.5 ng/ml to 1000 ng/ml, with an average regression coefficient of 0.999 (n=5) for both compounds. The lowest limit of quantitation was 25 ng/ml, with a signal-to-noise ratio of 8:1. The within-day relative standard deviations for O6BG quality control samples (n=18) with concentrations of 735 ng/ml, 305 ng/ml and 38 ng/ml were 2.4%, 4.2% and 5.3%, respectively. The within-day relative standard deviations for 8-oxo-O6BG quality control samples (n=18) at concentrations of 735 ng/ml, 420 ng/ml and 42 ng/ml were 2.2%, 4.0% and 7.1%, respectively. The day-to-day relative standard deviations for the same control specimens were 3.1%, 4.8% and 7.1% for O6BG, respectively, and 2.3%, 4.7% and 11.0% for 8-oxo-O6BG, respectively. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine. O6-Benzyl-8-oxo-guanine was identified in patient plasma specimens by liquid chromatography-electrospray mass spectrometry by comparison with spectral data acquired from reference material.

Antineoplastic Agents↗

PubMed1997

Selection for G156A O6-methylguanine DNA methyltransferase gene-transduced hematopoietic progenitors and protection from lethality in mice treated with O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea.

A retroviral gene therapy approach was developed to protect early hematopoietic progenitors from 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a stem cell toxin, and O6-benzylguanine (BG), an inhibitor of a key BCNU resistance protein, O6-alkylguanine DNA alkyltransferase (AGT). The retroviral vector MFG was used to transfer the G156A MGMT (deltaMGMT) cDNA, encoding a mutant AGT that is resistant to inhibition by BG, into murine bone marrow-derived hematopoietic progenitors. Following transplantation into lethally irradiated mice, the transduced cells were subjected to in vivo BG and BCNU treatment to examine the ability to enrich for transduced cells expressing deltaAGT. Transplantation of deltaMGMT-transduced cells resulted in deltaAGT expression in 30% of bone marrow nucleated cells 13 weeks after transplantation. After one cycle of BG and BCNU, deltaAGT expression was observed in 60% of bone marrow cells, and the percentage of colony-forming units (culture; CFU-C) containing proviral sequence increased from 67 to 100%. CFU-C obtained from BG and BCNU-treated deltaMGMT animals up to 23 weeks after transplantation were more resistant to combination BG and BCNU than CFU-C from mice transplanted with lacZ-transduced cells and treated with BG and BCNU or from mice transplanted with deltaMGMT-transduced cells and left untreated. The degree of drug resistance in deltaMGMT-transduced hematopoietic progenitors to BG and BCNU was much greater than we observed previously with wild-type MGMT gene transfer and treatment with BCNU alone. Furthermore, whereas 21 of 22 mice transplanted with deltaMGMT-transduced cells survived in vivo BG and BCNU administration, only 3 of 13 mice transplanted with lacZ-transduced progenitors survived similar drug treatment. Thus, deltaMGMT-transduced murine bone marrow cells selectively survive in vivo BG and BCNU exposure, resulting in prolonged enrichment for the transduced cells and protection from mortality induced by this drug combination.

Animals↗

PubMed1997

O6-methylguanine-DNA methyltransferase (MGMT) transfectants of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive colon cancer cell line selectively repopulate heterogenous MGMT+/MGMT- xenografts after BCNU and O6-benzylguanine plus BCNU.

To evaluate the role of O6-alkylguanine-DNA alkyltransferase (AGT) in colon tumor chloroethylnitrosourea (CENU) resistance, AGT-deficient VACO 8 cells were transfected with a vector containing or lacking the human O6-methylguanine-DNA methyltransferase (MGMT) cDNA. VACO 8MGMT (V8MGMT) sublines possessed high levels of AGT activity in cell culture and were > 10-fold resistant to the CENU 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). V8MGMT cells, VACO 8neo cells, and mixtures of both were grown as xenografts in nude mice. MGMT expression in VACO 8 xenografts reflected the percentage of V8MGMT cells present in the tumor inoculum. Xenografts originally containing 0-10% V8MGMT cells were sensitive to BCNU, although partial resistance was observed as the percentage of V8MGMT cells increased. Tumors containing 30-100% V8MGMT cells were completely resistant to BCNU with no regressions and no growth delays. Pretreatment with O6-benzylguanine (BG) depleted tumor AGT activity for at least 6 h and sensitized xenografts containing 1 and 100% V8MGMT cells to BCNU. After BCNU or BG + BCNU, xenografts growing from inoculums containing as low as 0.1% V8MGMT cells had high AGT activities similar to that found in V8MGMT xenografts, with the majority of the cells expressing MGMT. These results provide evidence that MGMT expression influences both intrinsic and acquired colon tumor CENU resistance, that selective expansion of AGT+ colon tumor cells commonly occurs after CENU exposure, and that BG is effective in sensitizing colon tumors to CENUs, even when only a small fraction of the cells in a heterogeneous tumor express MGMT.

Animals↗

PubMed1997

Potentiation of lymphomagenesis by methylnitrosourea in mice transgenic for LMO1 is blocked by O6-alkylguanine DNA-alkyltransferase.

We evaluated induction of lymphomas by the methylating carcinogen, N-methylnitrosourea [MNU], in transgenic mice expressing both LMO1 and the DNA repair gene, MGMT, in the thymus. The goal was to determine whether environmental mutagens shorten the latency or increase the incidence of LMO1 + lymphomas and whether mice transgenic for both LMO1 and MGMT, and thereby able to repair O6-methylguanine DNA adducts induced by MNU, would be protected. Mice heterozygous for LMO1 or MGMT were crossed and offspring treated with MNU at 6 weeks of age. MNU induced lymphoma incidence was highest in the LMO1 mice, 91% and lowest in the hMGMT + mice, 15%. MNU induced K-ras mutations in codon 12 in non-MGMT transgenics resulted in a shorter latency of tumors and accounting for half of the early lymphomas in LMO1 mice. The effect of MNU was abrogated in the LMO1/hMGMT transgenic mice, indicating the ability of MGMT expression to block the carcinogenic effect of MNU even in cancer prone mice. Thus, methylating agents potentiate lymphomagenesis of LMO1, in part through activation of K-ras and the MAPK pathway, a process which appear to synergize with LMO1 mediated transcription activation. O6-alkylguanine DNA-alkyltransferase mediated DNA repair effectively blocks chemical carcinogenesis in mice carrying the LMO1 oncogene.

Animals↗

PubMed1997

LacZ and interleukin-3 expression in vivo after retroviral transduction of marrow-derived human osteogenic mesenchymal progenitors.

Human marrow-derived mesenchymal progenitor cells (hMPCs), which have the capacity for osteogenic and marrow stromal differentiation, were transduced with the myeloproliferative sarcoma virus (MPSV)-based retrovirus, vM5LacZ, that contains the LacZ and neo genes. Stable transduction and gene expression occurred in 18% of cells. After culture expansion and selection in G418, approximately 70% of neo(r) hMPCs co-expressed LacZ. G418-selected hMPC retain their osteogenic potential and form bone in vivo when seeded into porous calcium phosphate ceramic cubes implanted subcutaneously into SCID mice. LacZ expression was evident within osteoblasts and osteocytes in bone developing within the ceramics 6 and 9 weeks after implantation. Likewise, hMPCs transduced with human interleukin-3 (hIL-3) cDNA, adhered to ceramic cubes and implanted into SCID mice, formed bone and secreted detectable levels of hIL-3 into the systemic circulation for at least 12 weeks. These data indicate that genetically transduced, culture-expanded bone marrow-derived hMPCs retain a precursor phenotype and maintain similar levels of transgene expression during osteogenic lineage commitment and differentiation in vivo. Because MPCs have been shown to differentiate into bone, cartilage, and tendon, these cells may be a useful target for gene therapy.

Adult↗

PubMed1997

Erythroid burst-forming units (BFU-E) predict hematopoietic recovery after peripheral blood progenitor cell transplantation in patients with advanced breast cancer.

In 29 consecutive heavily pretreated stage IV breast cancer patients, we analyzed patient factors and in vitro characteristics of peripheral blood progenitor cell (PBPC) collections that might correlate with the speed of hematopoietic recovery after autologous transplantation. PBPC collections were assessed for total number of mononuclear cells infused/patient weight in kg and hematopoietic progenitor cell content using in vitro colony-forming assays. In these patients, who received PBPC as the sole hematopoietic support after myeloablative chemotherapy, the number of erythroid burst-forming units (BFU-E) infused correlated significantly with both time to neutrophil (P = 0.008) and platelet (P = 0.0001) recovery and was a better predictor of hematopoietic recovery than number of CFU-GM administered. By day 75 after transplantation, six patients with poor BFU-E yields failed to engraft platelets. Our data suggest that the number of BFU-E infused correlate with time to hematopoietic engraftment and may predict failure of platelet engraftment in heavily pre-treated patients.

Adult↗

PubMed1997

Human bone marrow-derived mesenchymal (stromal) progenitor cells (MPCs) cannot be recovered from peripheral blood progenitor cell collections.

The purpose of this study was to compare the ability to collect human bone marrow-derived mesenchymal (stromal) progenitor cells (MPC) from bone marrow versus peripheral blood hematopoietic progenitor cell (PBPC) collections using in vitro and in vivo assays. Ten milliliter samples of PBPC collections mobilized from 11 patients undergoing autotransplants using chemotherapy followed by G-CSF 5-10 micrograms/kg were evaluated using in vitro and in vivo assays for hematopoietic progenitors and MPCs. Additionally, 10 ml samples of unstimulated bone marrow aspirates as well as PBPC collected after mobilization using G-CSF 10 micrograms/kg obtained from 3 normal, histocompatible allogeneic donors were analyzed for hematopoietic progenitors and MPCs. The MPCs were isolated and culture-expanded as adherent cells in vitro and subsequently tested for the capacity to differentiate into mesenchymal phenotypes in vivo using calcium hydroxyapatite porous ceramic cubes implanted s.c. in athymic mice. Demineralized sections of these cubes were analyzed histologically for the appearance of bone and cartilage. Seven autotransplant subjects with cancer received G-CSF after chemotherapy administration, whereas 4 cancer patients and all 3 normal donors received G-CSF alone as the mobilizing regimen. For the autologous PBPC collections and the normal marrow aspirations, median hematopoietic progenitor content was in the normal range for our institution. MPCs were detected in in vitro cultures and as bone-positive ceramic cubes in samples of all 3 allogeneic donor bone marrows but in none of the 14 autologous and 6 allogeneic PBPC collections. In conclusion, MPCs could not be recovered in PBPC collections obtained from either normal donors or patients who underwent PBPC collections after mobilization therapy but could be obtained routinely from bone marrow samples. Although the role of transplanted MPCs is an area of clinical investigation, this study points out a fundamental differences in the population of cells transplanted after collection from bone marrow versus peripheral blood.

Adult↗

PubMed1997

Human alkyltransferase-transduced murine myeloid progenitors are enriched in vivo by BCNU treatment of transplanted mice.

Retroviral gene transfer into murine hematopoietic progenitors of the human O6alkylguanine-DNA alkyltransferase cDNA, methylguanine methyltransferase (MGMT) has been shown to result in MGMT expression, increased alkyltransferase (AGT) activity, and resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) both in vitro and in vivo. In the present study we show that MGMT expressing bone marrow (BM) progenitors can be selected for in vivo by BCNU administration. MGMT+ mice treated with multiple doses of BCNU and examined 13 to 17 weeks after transplantation displayed a 2.4-fold increase in the percentage of progenitors with evidence of proviral integration (p < 0.0001). Likewise, percentage of the BCNU IC50 in these progenitors was 1.8-fold higher than that observed in progenitors of MGMT+ mice not treated with BCNU (p = 0.0027) and 3.6-fold higher than in mock-transduced progenitors (p < 0.001). AGT expression in myeloid cells was 3.8-fold higher in mice treated with BCNU than in untreated mice (p = 0.0378) and 64-fold higher than endogenous AGT levels. These findings demonstrate that after transplantation with MGMT-transduced BM cells, BCNU treatment enriches for MGMT+ cells, resulting in an increase in MGMT expression and AGT activity in vivo. This approach may be used to enrich for transduced hematopoietic cells in patients after clinical transplantation, to decrease myelosuppression after repeated nitrosourea exposure, and to increase the proportion of genetically altered hematopoietic cells.

Animals↗

PubMed1996

Mismatch repair mutations override alkyltransferase in conferring resistance to temozolomide but not to 1,3-bis(2-chloroethyl)nitrosourea.

Cells with the mutator phenotype are tolerant to methylating damage from N-methylnitrosourea and N-methyl-N'-nitro-N-nitrosoguanine, exhibit replication repair errors, and have recently been found to be mutant in mismatch repair (MMR). However, resistance of cell lines with these defects to clinically used chemotherapeutic agents and the relationship of this resistance to expression of O6-alkylguanine-DNA alkyltransferase (AGT), which repairs DNA damage caused by methylating agents, has not been demonstrated. We compared resistance to the methylating agent temozolomide (TMZ) and to the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), with and without AGT inhibition by 06-bG in several colorectal carcinoma cell lines. Two cell lines had known microsatellite instability (replication repair error-positive) and high levels of AGT, as well as a mutation in one of two MMR genes, hMLH1 (HCT116) or GTBP (HCT15). Cell line SW480 had wild-type MMR genes and high AGT, and HCT116+Ch3 has previously been transduced with chromosome 3 (carrying wild-type hMLH1) and thus has a "corrected" MMR phenotype. SW480 exhibited the expected sensitivity to TMZ and BCNU and marked potentiation of cytotoxicity by O6-bG. In contrast, HCT15 and HCT116 were markedly resistant to TMZ and were not sensitized by O6-bG-mediated inhibition of AGT, whereas the sensitivity pattern in HCT116+Ch3 cells was similar to that in SW480. All cell lines were sensitized to BCNU by O6-bG. Thus, tumor cells with defects in MMR appear particularly resistant to methylating agents in a manner that overrides dependence on AGT and its inhibition by O6-bG. However, these cells use AGT for resistance to chloroethylating agents, providing an alternative strategy for alkylating agent therapy.

Antineoplastic Agents, Alkylating↗

PubMed1996

Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to O6-benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea.

Human CD34 cells express low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Gene transfer of the AGT gene, methylguanine DNA methyltransferase (MGMT), results in only modest BCNU resistance. Recently, an AGT inhibitor, O6-benzylguanine (BG), entered clinical trials. In preclinical studies, BG potentiated the cytotoxic effect of BCNU in tumors but increased toxicity to normal CD34 cells. We transferred a mutant MGMT containing a glycine-to-alanine mutation at position 156, resulting in marked resistance to BG, into Chinese hamster cells; the K562 cell line and human CD34 cells used the retroviral backbone MFG. In each instance, cells expressed increased AGT and were much more resistant to the combination of BG and BCNU than the parental cells or cells transduced with wild-type MGMT. Furthermore, the transduction efficiency in human CD34 cells was in excess of 70%, and the proportion of CD34 transduced cells resistant to the combination was > 30%. Thus, retroviral-mediated transduction of a mutant MGMT into CD34 cells appears to be an effective way to induce selective resistance to a drug combination designed to overcome a significant resistance mechanism to nitrosoureas in tumors.

Animals↗

PubMed1996

Human CD34+ hematopoietic progenitors have low, cytokine-unresponsive O6-alkylguanine-DNA alkyltransferase and are sensitive to O6-benzylguanine plus BCNU.

Human bone marrow (BM) cells contain low levels of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase, which may explain their susceptibility to nitrosourea-induced cytotoxicity and the development of secondary leukemia after nitrosourea treatment. Isolated CD34+ myeloid progenitors were also found to have low levels of alkyltransferase activity. The level of alkyltransferase in CD34+ cells or in mononuclear BM cells did not increase after incubation with granulocyte-macrophage colony-stimulating factor, interleukin-3, stem cell factor, the combination, or 5637 conditioned medium. BCNU sensitivity remained unchanged as well. In addition, O6-benzylguanine depleted alkyltransferase activity in BM cells at concentrations as low as 1.5 mumol/L after a 1-hour exposure. O6-benzylguanine pretreatment markedly sensitized hematopoietic progenitor colony-forming cells to BCNU, resulting in a reduction in the dose of drug (termed the dose-modification factor) required to inhibit 50% of the colony formation (IC50) of threefold to fivefold. Since, unlike many other cell types, proliferating early (CD34+) hematopoietic precursors do not induce alkyltransferase, myelosuppression may be the dose-limiting toxicity of the combination of O6-benzylguanine plus BCNU in clinical trials.

Adult↗

PubMed1996

Determination of O6-benzylguanine in human plasma by reversed-phase high-performance liquid chromatography.

A high-performance liquid chromatographic assay for O6-benzylguanine utilizing liquid-liquid extraction and reversed-phase chromatography has been developed. Plasma samples were alkalinized, extracted into ethyl acetate, evaporated, and the residues were reconstituted and chromatographed. Separation was accomplished by gradient elution with a mobile phase of methanol, acetonitrile, and phosphate buffer, pH 3.2. Eluted compounds were detected spectrophotometrically at 280 nm. Sample quantitation was obtained from the regression line of six-point standard curves ranging from 25 to 400 ng/ml. O6-Benzylguanine peak heights were compared to peak heights of O6-(p-chlorobenzyl)guanine (internal standard). The average regression coefficient was 0.999 (n = 4). High concentration (305 ng/ml) and low concentration (38 ng/ml) quality control samples were determined with a day-to-day relative standard deviation of 7 and 8%, respectively (n = 18). The within-day relative standard deviations were 2.7 and 3.0% (n = 18) for the high and low concentration quality control specimens, respectively. Sample quantitation was reliable to 25 ng/ml with a signal-to-noise ratio of 8:1. This method was applied to plasma samples obtained from patients in a clinical trial of O6-benzylguanine.

Acetonitriles↗

PubMed1996

Differential sensitivity of human and mouse alkyltransferase to O6-benzylguanine using a transgenic model.

O6-benzlguanine (O6-bG) potentiates nitrosourea cytotoxicity of human tumor xenografts in nude mice by inactivating O6-alkylguanine-DNA alkyltransferase (AGT). Recent reports dispute whether murine AGT, in cell-free systems, is less sensitive to O6-bG than the human AGT protein, raising the possibility that efficacy seen in the mouse host may not predict the therapeutic index observed in clinical trials. To establish whether mouse and human AGT have different sensitivity to O(6)-bG, we evaluated in vitro and in vivo models of O(6)-methylguanine-DNA methyltransferase gene (MGMT) expression in the same genetic background. The 50% inhibitory concentration of O6-bG for inactivation of mouse AGT was >10-fold higher than for the human protein in MGMT-transfected Chinese hamster ovary (CHO) cells. A dose of O6-bG, which inactivated human AGT, markedly sensitized human MGMT-transfected CHO cells to 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU), whereas mouse MGMT-transfected CHO cells were much more resistant. O6-bG inactivation of AGT in vivo was studied in livers of human MGMT-transgenic mice expressing both human and mouse AGT. After a single dose of O6-bG i.p., the 50% inhibitory concentration of AGT was higher for mouse than for human AGT. To reconcile our finding with those of others, we sequenced the mouse MGMT cDNA and found that mutation of amino acid residue Leu180 was associated with O6-bG resistance. These studies provide strong evidence that inactivation of AGT both in vivo and in vitro by O6-bG is species selective and impacts O6-bG-mediated enhancement of BCNU toxicity. This may influence the therapeutic index of O6-bG-BCNU combinations observed in human tumor xenograft-bearing mice.

Animals↗

PubMed1996

Thymidine kinase deficient cells with decreased TTP pools are hypersensitive to DNA alkylating agents.

The effect of mutational loss of thymidine kinase (TK) on the sensitivity to alkylating agents was investigated in promyelocytic, HL-60, and T-lymphoblastoid, Molt-3, human leukemia cell lines. Although both cell lines exhibited approx. 1% residual TK activity, only HL-60 TK deficient cells had a decreased intracellular TTP pool, i.e., 20% of that of the wild-type. When treated with N-methyl-N'-nitronitrosoguanidine or ethyl methanesulfonate, HL-60 TK deficient cells showed significantly increased killing and mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus relative than did wild-type. Pretreatment of cells with O6-benzylguanine, an inhibitor of O6-alkylguanine-DNA alkyltransferase, partially abolished those differences. Molt-3 wild-type and TK deficient cells had similar cell survivals and HGPRT mutation frequencies following treatment with alkylating agents. These results indicate that TK deficiency, only when a concomitant decrease of TTP pool is detected, plays a pivotal role in the sensitivity to the cytotoxic and mutagenic effects of alkylating agents.

Antineoplastic Agents↗

PubMed1996

Role of DNA repair in resistance to drugs that alkylate O6 of guanine.

The mechanism of cytotoxicity of a number of chemotherapeutic agents involves alkylation at the O6 position of guanine, a site that strongly influences cytotoxicity. Repair of these lesions by the alkyltransferase protects from cytotoxicity and is a major mechanism of resistance to these agents. O6-benzylguanine inhibition of alkyltransferase sensitizes tumor cells, and clinical trials are underway to determine its efficacy. The use of gene therapy to enhance the expression of alkyltransferase in hematopoietic cells may prevent dose-limiting myelosuppression and may enhance the utility of this class of chemotherapeutic agents.

Amino Acid Sequence↗