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S L Gerson

Publications and source records attributed to S L Gerson.

At least 37 records · Page 2Linked to original sources
PubMed2000

Characterization of MLH1 and MSH2 DNA mismatch repair proteins in cell lines of the NCI anticancer drug screen.

PURPOSE AND METHODS: The lack of a functional DNA mismatch repair (MMR) pathway has been recognized as a common characteristic of several different types of human cancers due to mutation affecting one of the MMR genes or due to promoter methylation gene silencing. These MMR-deficient cancers are frequently resistant to alkylating agent chemotherapy such as DNA-methylating or platinum-containing compounds. To correlate drug resistance with MMR status in a large panel of human tumor cell lines, we evaluated by Western blot the cellular levels of the two MMR proteins most commonly mutated in human cancers, MLH1 and MSH2, in the NCI human tumor cell line panel. This panel consists of 60 cell lines distributed among nine different neoplastic diseases. RESULTS: We found that in most of these cell lines both MLH1 and MSH2 were expressed, although at variable levels. Five cell lines (leukemia CCRF-CEM, colon HCT 116 and KM12 and ovarian cancers SK-OV-3 and IGROV-1) showed complete deficiency in MLH1 protein. MSH2 protein was detected in all 57 cell lines studied. Absence of MLH1 protein was always linked to resistance to the methylating chemotherapeutic agent temozolomide. This resistance was independent of cellular levels of O6-alkylguanine DNA alkyltransferase. Based on data available for review in the NCI COMPARE database, cellular levels of MLH1 and MSH2 did not correlate significantly with sensitivity to any standard anticancer drug or with any characterized molecular target already tested against the same panel of cell lines. CONCLUSION: Based on evaluation of 60 tumor cell lines in the NCI anticancer drug screen, MLH1 deficiency was more common than MSH2 deficiency and was always associated with a high degree of temozolomide resistance. These data will enable correlations with other drug sensitivities and molecular targets in the COMPARE database to evaluate linked processes in tumor drug resistance.

Adaptor Proteins, Signal Transducing↗
PubMed2000

CD34+ selection of hematopoietic blood cell collections and autotransplantation in lymphoma: overnight storage of cells at 4 degrees C does not affect outcome.

The purpose of this study was to investigate whether storing mobilized peripheral blood progenitor cell (PBPC) collections overnight before CD34+ selection may delay platelet count recovery after high-dose chemotherapy and CD34+-enriched PBPC re-infusion. Lymphoma patients underwent PBPC mobilization with cyclophosphamide 4 g/m2 i.v. and G-CSF 10 microg/kg/day subcutaneously. Patients were prospectively randomized to have each PBPC collection enriched for CD34+ cells with the CellPro CEPRATE SC System either immediately or after overnight storage at 4 degrees C. Thirty-four patients were randomized to overnight storage and 34 to immediate processing of PBPC; 15 were excluded from analysis due to tumor progression or inadequate CD34+ cell mobilization. PBPC from 23 patients were stored overnight, while 30 subjects underwent immediate CD34+ selection and cryopreservation. Median yield of CD34+ enrichment was 43.6% in the immediate processing group compared to 39.1% in the overnight storage group (P = 0.339). Neutrophil recovery >500 x 10(9)/l occurred a median of 11 days (range 9-16 days) in the overnight storage group compared to 10.5 days (range 9-21 days) in the immediate processing group (P = 0.421). Median day to platelet transfusion independence was 13 (range 7-43) days in the overnight storage group vs 13.5 (range 8-35) days in those assigned to immediate processing (P = 0.933). We conclude that storage of PBPC overnight at 4 degrees C allows pooling of consecutive-day collections resulting in decreased costs and processing time without compromising neutrophil and platelet engraftment after infusion of CD34+-selected progenitor cells. Bone Marrow Transplantation(2000) 25, 559-566.

Adolescent↗
PubMed2000

Human marrow-derived mesenchymal stem cells (MSCs) express hematopoietic cytokines and support long-term hematopoiesis when differentiated toward stromal and osteogenic lineages.

Human mesenchymal stem cells (MSCs), bone marrow-derived pluripotent adherent cells of mesenchymal origin can differentiate along the osteogenic, chondrogenic, adipogenic, and tendonogenic lineages. In this report we characterize cytokine and growth factor gene expression by MSCs and investigate the modulation of cytokine expression that occurs during osteogenic and stromal differentiation. MSCs constitutively expressed mRNA for interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), and stem cell factor (SCF). MSCs treated with IL-1alpha upregulated mRNA levels of IL-6, IL-11, and LIF, and began to express detectable levels of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF). mRNA levels of M-CSF and SCF did not change. MSCs cultured in osteogenic medium differentiated along the osteogenic lineage and downregulated mRNA levels of IL-6, IL-11 and LIF whereas, M-CSF and SCF expression were unchanged and G-CSF and GM-CSF remained undetectable. IL-3 was not detected in MSC culture under any conditions. MSCs precultured in control medium, IL-1alpha, or osteogenic medium maintained similar capacity to support long-term culture initiating cell (LT-CIC). Thus, primary and osteogenic differentiated MSCs produce important hematopoietic cytokines and support hematopoiesis in long-term cultures, suggesting that these cells may provide an excellent ex vivo environment for hematopoiesis during progenitor cell expansion and may be important for in vivo cell therapy.

Bone Marrow Cells↗
PubMed2000

Heterozygous DNA mismatch repair gene PMS2-knockout mice are susceptible to intestinal tumor induction with N-methyl-N-nitrosourea.

PMS2-deficient (PMS2(-/-)) mice are hypersensitive to N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas based on the failure to initiate mismatch repair (MMR) at O(6)-methylguanine:T mismatches formed after MNU exposure. However, heterozygous PMS2 knockout (PMS2(+/-)) mice do not develop spontaneous tumors, suggesting that they have sufficient MMR function to prevent genomic instability. We hypothesized that in PMS2(+/-) mice, exogenous carcinogens may either mutationally knockout the remaining normal allele leading cells to develop tumors or introduce sufficient DNA adducts and mismatches to overload the lower capacity for MMR, leading in either case to an increased rate of tumor production. In the present study, PMS2(+/-) mice and their littermate PMS2(+/+) mice were monitored for tumor incidence following MNU treatment. Mice were given 50 mg MNU/kg i.p. when 5 weeks old. They demonstrated a similar incidence of thymic lymphomas, suggesting that expression of the single normal PMS2 allele is sufficient to protect the thymus and implying that a single dose of MNU may not efficiently knock out the remaining PMS2 allele in the thymus. Surprisingly, PMS2(+/-) mice were significantly more likely to develop intestinal tumors-both adenomas and adenocarcinomas-after MNU than were PMS2(+/+) mice (2.34 +/- 0.34 tumors per mouse versus 1.34 +/- 0.25 tumors per mouse; P < 0.05). The intestinal tumors were located mainly in the small intestine. However, these tumors in both the PMS2(+/-) mice and PMS2(+/+) mice did not show microsatellite instability characteristic of loss of MMR. These results suggest that a single normal PMS2 allele can protect thymus but not intestine from MNU carcinogenesis. Organ-specific factors might influence MMR- mediated resistance to methylating agents. Heterozygous PMS2 knockout mice may be used as a promising animal model for intestinal tumorigenesis studies involving environmental carcinogens.

Adenosine Triphosphatases↗
PubMed2000

Impaired growth and elevated fas receptor expression in PIGA(+) stem cells in primary paroxysmal nocturnal hemoglobinuria.

The genetic defect underlying paroxysmal nocturnal hemoglobinuria (PNH) has been shown to reside in PIGA, a gene that encodes an element required for the first step in glycophosphatidylinositol anchor assembly. Why PIGA-mutated cells are able to expand in PNH marrow, however, is as yet unclear. To address this question, we compared the growth of affected CD59(-)CD34(+) and unaffected CD59(+)CD34(+) cells from patients with that of normal CD59(+)CD34(+) cells in liquid culture. One hundred FACS-sorted cells were added per well into microtiter plates, and after 11 days at 37 degrees C the progeny were counted and were analyzed for their differentiation pattern. We found that CD59(-)CD34(+) cells from PNH patients proliferated to levels approaching those of normal cells, but that CD59(+)CD34(+) cells from the patients gave rise to 20- to 140-fold fewer cells. Prior to sorting, the patients' CD59(-) and CD59(+)CD34(+) cells were equivalent with respect to early differentiation markers, and following culture, the CD45 differentiation patterns were identical to those of control CD34(+) cells. Further analyses of the unsorted CD59(+)CD34(+) population, however, showed elevated levels of Fas receptor. Addition of agonist anti-Fas mAb to cultures reduced the CD59(+)CD34(+) cell yield by up to 78% but had a minimal effect on the CD59(-)CD34(+) cells, whereas antagonist anti-Fas mAb enhanced the yield by up to 250%. These results suggest that expansion of PIGA-mutated cells in PNH marrow is due to a growth defect in nonmutated cells, and that greater susceptibility to apoptosis is one factor involved in the growth impairment.

Antigens, CD34↗
PubMed2000

Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy.

PURPOSE: Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We hypothesized that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by hematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of culture-expanded MSCs in breast cancer patients receiving autologous peripheral-blood progenitor-cell (PBPC) infusion. PATIENTS AND METHODS: We developed an efficient method of isolating and culture-expanding a homogenous population of MSCs from a small marrow-aspirate sample obtained from 32 breast cancer patients. Twenty-eight patients were given high-dose chemotherapy and autologous PBPCs plus culture-expanded MSC infusion and daily granulocyte colony-stimulating factor. RESULTS: Human MSCs were successfully isolated from a mean +/- SD of 23.4 +/- 5.9 mL of bone marrow aspirate from all patients. Expansion cultures generated greater than 1 x 10(6) MSCs/kg for all patients over 20 to 50 days with a mean potential of 5.6 to 36.3 x 10(6) MSCs/kg after two to six passages, respectively. Twenty-eight patients were infused with 1 to 2.2 x 10(6) expanded autologous MSCs/kg intravenously over 15 minutes. There were no toxicities related to the infusion of MSCs. Clonogenic MSCs were detected in venous blood up to 1 hour after infusion in 13 of 21 patients (62%). Median time to achieve a neutrophil count greater than 500/microL and platelet count >/= 20,000/microL untransfused was 8 days (range, 6 to 11 days) and 8.5 days (range, 4 to 19 days), respectively. CONCLUSION: This report is the first describing infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed rapid hematopoietic recovery suggests that MSC infusion after myeloablative therapy may have a positive impact on hematopoiesis and should be tested in randomized trials.

Adult↗
PubMed2000

Randomized cross-over trial of progenitor-cell mobilization: high-dose cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) versus granulocyte-macrophage colony-stimulating factor plus G-CSF.

PURPOSE: Patient response to hematopoietic progenitor-cell mobilizing regimens seems to vary considerably, making comparison between regimens difficult. To eliminate this inter-patient variability, we designed a cross-over trial and prospectively compared the number of progenitors mobilized into blood after granulocyte-macrophage colony-stimulating factor (GM-CSF) days 1 to 12 plus granulocyte colony-stimulating factor (G-CSF) days 7 to 12 (regimen G) with the number of progenitors after cyclophosphamide plus G-CSF days 3 to 14 (regimen C) in the same patient. PATIENTS AND METHODS: Twenty-nine patients were randomized to receive either regimen G or C first (G1 and C1, respectively) and underwent two leukaphereses. After a washout period, patients were then crossed over to the alternate regimen (C2 and G2, respectively) and underwent two additional leukaphereses. The hematopoietic progenitor-cell content of each collection was determined. In addition, toxicity and charges were tracked. RESULTS: Regimen C (n = 50) resulted in mobilization of more CD34(+) cells (2.7-fold/kg/apheresis), erythroid burst-forming units (1.8-fold/kg/apheresis), and colony-forming units-granulocyte-macrophage (2.2-fold/kg/apheresis) compared with regimen G given to the same patients (n = 46; paired t test, P<.01 for all comparisons). Compared with regimen G, regimen C resulted in better mobilization, whether it was given first (P =.025) or second (P =.02). The ability to achieve a target collection of > or =2x10(6) CD34(+) cells/kg using two leukaphereses was 50% after G1 and 90% after C1. Three of the seven patients in whom mobilization was poor after G1 had > or =2x10(6) CD34(+) cells/kg with two leukaphereses after C2. In contrast, when regimen G was given second (G2), seven out of 10 patients failed to achieve the target CD34(+) cell dose despite adequate collections after C1. Thirty percent of the patients (nine of 29) given regimen C were admitted to the hospital because of neutropenic fever for a median duration of 4 days (range, 2 to 10 days). The higher cost of regimen C was balanced by higher CD34(+) cell yield, resulting in equivalent charges based on cost per CD34(+) cell collected. CONCLUSION: We report the first clinical trial that used a cross-over design showing that high-dose cyclophosphamide plus G-CSF results in mobilization of more progenitors then GM-CSF plus G-CSF when tested in the same patient regardless of sequence of administration, although the regimen is associated with greater morbidity. Patients who fail to achieve adequate mobilization after regimen G can be treated with regimen C as an effective salvage regimen, whereas patients who fail regimen C are unlikely to benefit from subsequent treatment with regimen G. The cross-over design allowed detection of significant differences between regimens in a small cohort of patients and should be considered in design of future comparisons of mobilization regimens.

Adolescent↗
PubMed1999

Characterization of the P140K, PVP(138-140)MLK, and G156A O6-methylguanine-DNA methyltransferase mutants: implications for drug resistance gene therapy.

The G156A O6-alkylguanine-DNA alkyltransferase (AGT) mutant protein, encoded by the G156A O6-methylguanine-DNA methyltransferase gene (MGMT), is resistant to O6-benzylguanine (BG) inactivation and, after transduction into hematopoietic progenitors, transmits remarkable resistance to BG and BCNU. As a result, a clinical trial, in which the MGMT gene is transduced into CD34+ cells of patients with cancer, has been approved. A newly identified AGT mutation, P140K, generates dramatically increased BG resistance relative to G156A, and suggests that gene transfer of P140K may confer improved hematopoietic cell protection. To address this hypothesis, we measured BG + BCNU and BG + TMZ resistance in G156A, P140K, or P138M/V139L/P140K (MLK) MGMT-transduced K562 cells. In addition, we performed a detailed characterization of individual properties including BG resistance, activity, and protein stability of these mutants in human hematopoetic K562 cells and E86 retroviral producer cells. In K562 cell extracts, the MLK and P140K mutants retained full activity at doses up to 1 mM BG, while G156A had a BG ED50 of 15 microM, compared with 0.1 microM for wtAGT. In the absence of BG, the G156A protein possessed a 56% reduction in specific O6-methyltransferase activity compared with wtAGT. MLK, P140K, and wtAGT all possessed similar specific activities, although the O6-methyl repair rate of all mutants was reduced 4- to 13-fold relative to wtAGT. The wtAGT, MLK, and P140K proteins were stable, with half-lives of greater than 18 hr. In contrast, only 20% of the G156A protein was stable after 12 hr in cycloheximide and, interestingly, the remaining protein appeared to retain most of the activity present in non-cycloheximide-treated cells. Differences in BG resistance, activity, and stability between P140K, MLK, and G156A suggest that P140K may be the optimal mutant for drug resistance gene transfer. However, hematopoietic K562 cells transduced with MFG-G156A, P140K, or MLK had similar degrees of BG and BCNU as well as BG and TMZ resistance when treated with concentrations of BG (< or =25 microM) achieved in clinical trials, suggesting similar efficacy in many in vivo applications.

Antineoplastic Agents↗
PubMed1999

Mice defective in the DNA mismatch gene PMS2 are hypersensitive to MNU induced thymic lymphoma and are partially protected by transgenic expression of human MGMT.

DNA mismatch repair (MMR) stabilizes the cellular genome. Mice defective in the MMR gene PMS2 are susceptible to spontaneous thymic lymphoma and sarcomas. To determine the sensitivity of PMS2 knockout mice to environmental carcinogens and the protective effect of O6-methylguanine DNA methyltransferase (MGMT), heterozygous PMS2 knockout mice and human MGMT (hMGMT) transgenic mice were mated and the PMS2-/- and PMS2+/+ with or without hMGMT offspring were treated at 5 weeks of age with 50 mg/kg N-methyl-N-nitrosourea (MNU). MNU produces carcinogenic O6-methylguanine (O6-meG) adducts, resulting in thymic lymphoma in mice, which can be prevented in normal mice by overexpression of hMGMT. A significantly higher incidence of thymic lymphomas was observed in MNU-treated PMS2-/- mice, compared to wildtype PMS2+/+ mice (100 vs 52%; P < 0.001). The mean latency of lymphomas was also significantly shortened in PMS2-/- mice (81 vs 102 days, P < 0.01). Transgenic expression of hMGMT significantly but incompletely blocked MNU lymphomagenesis in PMS2-/- mice. The incidence of lymphomas in PMS2-/-/hMGMT+ mice was reduced to 80% (P < 0.01) and mean latency increased to 91 days (P < 0.05). Thymic lymphomagenesis was efficiently blocked in PMS2+/+/hMGMT+ mice with rapid repair of O6-meG. Since O6-meG:T mismatches in MMR+ cells may trigger mismatch repair resulting in abortive repair and cell death whereas in the absence of MMR, these mismatches are converted to A:T, we predicted that G to A point mutations in codon 12 of the K-ras gene would occur. In this study, we found G to A point mutations in codon 12 of the K-ras gene in many tumors. Thus, in MMR deficient tissues, methylating agents induce point mutations in cells with a higher rate of cell survival which together are potently carcinogenic in the thymus. These data suggest that PMS2 defective lymphomas may arise by the concerted action of environmental and perhaps endogenous methylation of DNA coupled to genomic instability.

Adenosine Triphosphatases↗
PubMed1999

Mice over-expressing human O6 alkylguanine-DNA alkyltransferase selectively reduce O6 methylguanine mediated carcinogenic mutations to threshold levels after N-methyl-N-nitrosourea.

While it is well known that MNU induces thymic lymphomas in the mouse, it remains unclear which pre-mutagenic lesions are responsible for lymphomagenic transformation. One lesion thought to play a critical role is O6methylguanine[O6mG]which initiates G: C to A:T transition mutations in K-ras and other oncogenes. O6alkylguanine-DNA alkyltransferase (AGT), encoded by the methylguanine methyltransferase gene [MGMT], removes the methyl group thereby preventing the mutation from occurring. When overexpressed in the thymus, MGMT protects mice from MNU-induced thymic lymphomas. To determine whether MGMT overexpression reduced G: C to A: T mutation frequency after MNU, Big Blue lacI and MGMT+/Big Blue mice were treated with MNU and analysed for mutations in the lacI and K-ras genes. The incidence of MNU-induced lymphomas was 84% in Big Blue lacI mice compared to 14% in MGMT+Big Blue lacI mice. Sixty-two per cent of the lymphomas had a GGT to GAT activating mutation in codon 12 of K-ras consistent with O6mG adduct-mediated point mutagenesis. LacI mutation frequency in thymus of MNU treated Big Blue mice was 45-fold above background whereas it was 11-fold above background in MNU treated MGMT+/Big Blue mice. Most lacI mutations were G:C to A:T transitions, implicating O6mG even in the MGMT+mice. No mutations were attributable to chromosomal aberrations or rearrangements. Thus, O6mG adducts account for the carcinogenic effect of MNU and MGMT overexpression is selectively able to reduce O6methylguanine adducts below a carcinogenic threshold. Other adducts are mutagenic but appear to contribute much less to malignant transformation or oncogene activation.

Alkylating Agents↗
PubMed1999

O6-benzylguanine: a clinical trial establishing the biochemical modulatory dose in tumor tissue for alkyltransferase-directed DNA repair.

Early phase evaluation of anticancer drugs has traditionally used toxicity (usually hematological) rather than efficacy end points to establish appropriate dosing schedules. To establish a biochemical efficacy end point for overcoming alkylguanine DNA alkyltransferase (AGT)-mediated tumor cell resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea, we performed a novel dose escalation clinical trial for the AGT-depleting agent O6-benzylguanine (BG). The dose of BG required to deplete AGT to undetectable levels (BMD(T)) in sequential computed tomography-guided tumor tissue biopsies before BG and 18 h after BG was determined. Thirty patients received doses of BG ranging from 10 to 120 mg/m2. In tumor tissue, AGT depletion >86% of baseline was demonstrated at all doses tested. Residual tumor AGT activity, present 18 h after BG doses of 10-80 mg/m2, was eliminated at the 120 mg/m2 dose and is thus the BMD(T) of BG. BG pharmacokinetics are characterized by the rapid, dose-independent clearance of BG from plasma Metabolism of BG to its biologically active metabolite, 8-oxo-benzylguanine (8-oxo-BG), was found. The t(1/2) of 8-oxo-BG is longer than BG. Plasma concentrations of 8-oxo-BG well above 200 ng/ml 18 h after the end of the BG infusion were observed at the highest dose levels tested and appeared to correlate with depletion of AGT activity to undetectable levels in tumor tissue. AGT activity in peripheral blood mononuclear cells at baseline did not correlate with tumor tissue AGT activity. Depletion of AGT activity to undetectable levels in peripheral blood mononuclear cells occurred at lower doses and was not a reliable predictor for tumor tissue depletion. No serious side effects were observed with administration of BG alone or in combination with 13 mg/m2 1,3-bis(2-chloroethyl)-1-nitrosourea. This is the first clinical study in which biochemical analyses from pre- and posttreatment tumor biopsies have been used as an efficacy end point for the clinical development of an anticancer agent. From our tumor tissue biopsy data, we have established that a BG dose of 120 mg/m2 infused over 1 h should be used in Phase II clinical trials.

Adult↗
PubMed1999

DeltaMGMT-transduced bone marrow infusion increases tolerance to O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea and allows intensive therapy of 1,3-bis(2-chloroethyl)-1-nitrosourea-resistant human colon cancer xenografts.

O6-Benzylguanine (BG) is a potent inhibitor of the DNA repair protein 06-alkylguanine DNA alkyltransferase (AGT), and sensitizes tumors to BCNU in vitro and in xenografts. The combination of BG and BCNU is now undergoing phase I clinical testing. The maximally tolerated dose of BCNU given after BG is expected to be lower then the doses tolerated as a single agent owing to BG sensitization of hematopoietic progenitors. We have previously shown that retroviral expression of G156A mutant MGMT (deltaMGMT) in mouse and human marrow cells results in significant BG and BCNU resistance. In this study we evaluated the effect of deltaMGMT-transduced marrow infusion on the therapeutic index of multiple BG and BCNU treatments in tumor-bearing nude (nu/nu athymic) mice. Prior to subcutaneous implantation of BCNU-resistant SW480 human colon cancer cells, cohorts of mice were given intraperitoneal injections of nonablative doses of BG (30 mg/kg) and BCNU (10 mg/kg, one-half of the LD10) and then infused with 1-2 x 10(6) isogeneic deltaMGMT (n = 29 mice) or lacZ-transduced (n = 20 mice) marrow cells. The xenograft-bearing mice were treated with multiple cycles of BG (30 mg/kg) and BCNU (10-25 mg/kg). After three cycles, deltaMGMT mouse bone marrow was repopulated with CFU containing the provirus, and demonstrated a 2.7-fold increase in AGT activity and a 5.5-fold increase in BCNU IC90 compared with LacZ mice. After five cycles, the BCNU IC90 of CFU cells increased nine-fold over control cells, indicating selective enrichment of CFU precursor cells expressing high levels of deltaMGMT. Starting with the third cycle of therapy, tolerance to BG and BCNU was significantly improved in deltaMGMT mice compared with LacZ mice, as evidenced by preserved peripheral blood counts, bone marrow cellularity, and CFU content 1 and 2 weeks posttreatment and a significantly higher survival rate. Xenograft growth was significantly delayed in mice tolerating multiple cycles and higher dose intensity of BG and BCNU as compared with mice receiving less intensive therapy. We conclude that deltaMGMT-transduced marrow cells can improve the therapeutic index of BG and BCNU by selectively repopulating the marrow and providing significant marrow tolerance to this combination, allowing intensive therapy of a BCNU-resistant tumor.

Animals↗
PubMed1999

Acquired resistance to O6-benzylguanine plus chloroethylnitrosoureas in human breast cancer.

PURPOSE: O(6)-benzylguanine (BG) is a pseudosubstrate inactivator of the DNA repair protein O(6)- alkylguanine-DNA alkyltransferase (AGT) that has entered clinical trials as a potentiator of the antitumor effect of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study was designed to evaluate potential mechanisms of BG + BCNU resistance in breast cancer cells. METHODS: We treated MCF-7 breast cancer cells three times with cytotoxic concentrations of BG + BCNU to isolate a population of MCF-7 cells possessing BG + BCNU resistance (BBR). We measured drug resistance, AGT activity, cross-resistance to other agents and sensitivity of AGT to BG inactivation. RESULTS: When compared with the parent line, MCF-7BBR cells were no longer sensitized to BCNU by BG, resulting in three-fold resistance to BG + BCNU and fourfold resistance to BG + 1-(2-chloroethyl)-1-nitrosourea (CNU). In contrast, MCF-7 and MCF-7BBR cells had similar sensitivity to BCNU, CNU, temozolomide (each in the absence of BG), cisplatin, and UV light. Acquired resistance to BG + chloroethylnitrosoureas (CENU) in MCF-7BBR cells was not accompanied by an increase in AGT or altered kinetics of BG inactivation of AGT. While glutathione-S transferase activity was increased modestly, its contribution to resistance in this setting remains unclear. CONCLUSIONS: This is the first report of acquired BG + CENU resistance in human tumor cells and the results indicate that selective pressure with BG + CENUs may result in nonresponsiveness to BG by one or more complex mechanisms. These results predict the development of acquired resistance to BG + BCNU in clinical trials.

Antineoplastic Agents, Alkylating↗
PubMed1999

Bone marrow-derived mesenchymal stem cells remain host-derived despite successful hematopoietic engraftment after allogeneic transplantation in patients with lysosomal and peroxisomal storage diseases.

Human bone marrow contains mesenchymal stem cells (MSCs) that can differentiate into various cells of mesenchymal origin. We developed an efficient method of isolating and culture expanding a homogenous population of MSCs from bone marrow and determined that MSCs express alpha-L-iduronidase, arylsulfatase-A and B, glucocerebrosidase, and adrenoleukodystrophy protein. These findings raised the possibility that MSCs may be useful in the treatment of storage disorders. To determine if donor derived MSCs are transferred to the recipients with lysosomal or peroxisomal storage diseases by allogeneic hematopoietic stem cell (HSC) transplantation, we investigated bone marrow derived MSCs of 13 patients 1-14 years after allogeneic transplantation. Highly purified MSCs were genotyped either by fluorescence in situ hybridization using probes for X and Y-chromosomes in gender mis-matched recipients or by radiolabeled PCR amplification of polymorphic simple sequence repeats. Phenotype was determined by the measurement of disease specific protein/enzyme activity in purified MSCs. We found that MSCs isolated from recipients of allogeneic HSC transplantation are not of donor genotype and have persistent phenotypic defects despite successful donor type hematopoietic engraftment. Whether culture expanded normal MSCs can be successfully transplanted into patients with storage diseases and provide therapeutic benefit needs to be determined.

Case-Control Studies↗
PubMed1999

Human mesenchymal stem cells provide stromal support for efficient CD34+ transduction.

Human mesenchymal stem cells (hMSC)-nonhematopoietic cells within the bone marrow microenvironment that can be culture expanded to a uniform population of fibroblastic cells-have been shown to support long-term hematopoiesis of CD34+ cells. Because direct contact between stromal elements and CD34+ cells enhances long-term engraftment, we postulated that hMSC would be a good alternative to the more heterogeneous stroma currently used in gene transfer studies. We used hMSC to support retroviral gene transfer of the G156A MGMT (deltaMGMT) gene encoding an alkyltransferase (AGT), which confers drug resistance to a combination of O6-benzylguanine (BG) plus the alkylating agents BCNU and temozolomide (TMZ) in human hematopoietic progenitors. In the presence of IL-3, IL-6, SCF, or leukemia inhibitory factor (LIF) and Flt-3 ligand, hMSC facilitated expansion and retroviral transduction of human peripheral blood-mobilized CD34+ cells. Furthermore, the transduced cells expressed AGT in 29% of hematopoietic cells and were 5-fold more resistant to BCNU and TMZ than were untransduced cells. Unirradiated hMSC present as support cells were simultaneously transduced and expressed AGT in 26% of the cells. Thus, the homogeneous nature of hMSC, and their ability to support gene transfer and be transduced themselves suggest they may be useful in clinical gene transfer protocols and have broad therapeutic applications.

Antigens, CD↗
PubMed1999

Reduced lung tumorigenesis in human methylguanine DNA--methyltransferase transgenic mice achieved by expression of transgene within the target cell.

Human methylguanine-DNA methyltransferase (MGMT) transgenic mice expressing high levels of O6-alkylguanine-DNA alkyltransferase (AGT) in lung were crossbred to A/J mice that are susceptible to pulmonary adenoma to study the impact of O6-methylguanine (O6mG)-DNA adduct repair on NNK-induced lung tumorigenesis. Expression of the chimeric human MGMT transgene in lung was identified by northern and western blot analysis, immunohistochemistry assay and enzymatic assay. AGT activity was 17.6 +/- 3.2 versus 1.2 +/- 0.4 fmol/microg DNA in lung of MGMT transgenic mice compared with non-transgenic mice. Immunohistochemical staining with anti-human AGT antibody showed that human AGT was expressed throughout the lung. However, some epithelial cells of bronchi and alveoli did not stain for human AGT, suggesting that the human MGMT transgene expression was heterogeneous. After 100 mg/kg NNK i.p. injection in MGMT transgenic mice, lung AGT activity remained much higher and levels of lung O6mG-DNA adducts in MGMT transgenic mice were lower than those of non-transgenic mice. In the tumorigenesis study, mice received 100 mg/kg NNK at 6 weeks of age and were killed 44 weeks later. Ten of 17 MGMT transgenic mice compared with 16 of 17 non-transgenic mice had lung tumors, P < 0.05. MGMT transgenic mice had lower multiplicity and smaller sized lung tumors than non-transgenic mice. Moreover, a reduction in the frequency of K-ras mutations in lung tumors was found in MGMT transgenic mice (6.7 versus 50% in non-transgenic mice). These results indicate that high levels of AGT expressed in mouse lung reduce lung tissue susceptibility to NNK-induced tumorigenesis due to increased repair capacity for O6mG, subsequently, decreased mutational activation of K-ras oncogene. Heterogeneity in the level of AGT expressed in different lung cell populations or other forms of carcinogenic DNA damage caused by NNK may explain the residual incidence of lung tumors in MGMT transgenic mice.

Adenoma↗