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Stanton L Gerson

Publications and source records attributed to Stanton L Gerson.

At least 37 records · Page 2Linked to original sources
PubMed2003

Repopulating defect of mismatch repair-deficient hematopoietic stem cells.

Mismatch repair deficiency is associated with carcinogenesis, increased spontaneous and induced mutagenesis, and resistance to methylating agents. In humans, leukemias and lymphomas arise in the background of mismatch repair deficiency, raising the possibility that hematopoiesis is abnormal as well. To address hematopoiesis in MSH2-/- mice, we collected marrow and performed serial transplantations of these cells, alone or mixed with wild-type cells, into lethally irradiated healthy mice. Transplant recipients were observed or treated with the methylating agent, temozolomide (TMZ). Methylating agent tolerance was evident by the competitive survival advantage of MSH2-/- marrow progenitors compared with wild-type cells after each TMZ exposure. However, serial repopulation by MSH2-/- cells was deficient compared with wild-type cells. In recipients of mixed populations, the MSH 2-/- cells were lost from the marrow, and mice receiving MSH2-/- cells plus TMZ could not be reconstituted in the third passage, whereas all wild-type cell recipients survived. No differences in telomere length, cell cycle distribution, or homing were observed, but an increase in microsatellite instability was seen in the MSH2-/- early progenitor colony-forming unit (CFU) and Sca+Kit+lin--derived clones. Thus, mismatch repair deficiency is associated with a hematopoietic repopulation defect and stem cell exhaustion because of accumulation of genomic instability.

Animals↗
PubMed2003

A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell lines.

BACKGROUND: Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. METHODS: We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O(6)-methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. RESULTS: EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34(+) cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O(6)-benzylguanine (BG)/N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. CONCLUSIONS: This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells.

Animals↗
PubMed2003

A phase I and pharmacodynamic study of sequential topotecan and etoposide in patients with relapsed or refractory acute myelogenous and lymphoblastic leukemia.

We designed a pharmacokinetic and pharmacodynamic phase I study of sequential topotecan (2.55-6.3mg/m2) by 72h infusion followed by five daily doses of etoposide for patients with refractory acute leukemia based upon synergistic anti-tumor activity of topoisomerase I and II inhibitors in vitro. Eight of the 29 patients achieved bone marrow aplasia and two patients achieved clinical remission. Common grade 3-4 toxicities included hepatic and gastrointestinal dysfunction, and correlated with increased steady-state plasma topotecan concentration. The predicted up-regulation of topoisomerase II activity by topoisomerase I inhibition was not observed at this dose and schedule and may provide insight into the modest anti-leukemia activity of the regimen.

Acute Disease↗
PubMed2003

Cytokines, including stem cell factor alone, enhance lentiviral transduction in nondividing human LTCIC and NOD/SCID repopulating cells.

Hematopoietic stem cells (HSC) require extensive cytokine-mediated stimulation and proliferation for efficient transduction by oncoretroviral vectors. Since lentiviral vectors can transduce nondividing cells, the need for cytokine stimulation has been questioned. We studied HIV-based lentiviral transduction of human early hematopoietic progenitors from umbilical cord blood in the presence or absence of IL-3, IL-6, stem cell factor (SCF), and Flt-3L (36SF) or SCF alone and characterized the effects of these conditions on the stem cell phenotype. Gene transfer was significantly higher in the presence of 36SF in mass culture cells, CFC, LTCIC, and NOD/SCID repopulating cells (SRC). Transduction of primitive progenitor/stem cells was poor without cytokines, with only 12% LTCIC and 23% SRC transduced, compared to 59% in LTCIC and 81% in SRC with 36SF. SCF alone matched transduction rates of multiple cytokines with 70% in CFC. Cytokines prevented apoptosis, expanded CD34(+) cell number, and maintained CFC and LTCIC frequencies. Cytokine stimulation increased transduction of nondividing Ara-C-resistant and aphidicolin-inhibited cells similar to dividing cells. These data suggest that cytokines enhance lentiviral transduction of HSC, without requiring cell division, and maintain the stem cell phenotype. SCF stimulation alone was sufficient for high level transduction.

Animals↗
PubMed2003

Myeloablation is not required to select and maintain expression of the drug-resistance gene, mutant MGMT, in primary and secondary recipients.

Gene transduction of hematopoietic progenitors capable of reconstituting both primary and secondary recipients is an important milestone in preclinical development of gene therapy. Myeloablation conditioning prior to infusion of transduced stem cells causes significant host morbidity. In contrast, drug-resistance gene transfer utilizes judicious in vivo selection of transduced stem cells over time, reaching only the level of transduction and expression required. The O(6)-benzylguanine (BG)-resistant mutant O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a potent selection gene for transduced cells. Using two different mutant MGMTs, G156A and P140K, that vary in BG resistance by a factor of 1:20, we asked whether long-term repopulating and secondary mouse-repopulating cells could be transduced, transplanted, and selected for in the nonmyeloablated recipient and whether the mutant MGMT would continue to be expressed in secondary recipient repopulating cells. We found that under stringent drug-selection competition, cells expressing the more BG-resistant variant, P140K-MGMT, were enriched over G156A-MGMT-expressing progenitors. In addition, the MFG retroviral vector transmitted the mutant MGMT gene to long-term repopulating cells that, after selective enrichment in the nonmyeloablated primary recipient, repopulated secondary mice and continued to express the transgene. Thus, MFG mutant MGMT vectors transduce repopulating hematopoietic stem cells that may be used both for chemotherapeutic drug resistance and to enrich for second therapeutic genes.

Animals↗
PubMed2003

Drug selection of mutant methylguanine methyltransferase from different oncoretroviral backbones results in multilineage hematopoietic transgene expression in primary and secondary recipients.

Optimized hematopoietic gene therapy requires vectors with strong expression in the desired target cell population and the ability to select for the expressing transduced cells. In the context of drug resistance selection of repopulating hematopoietic stem cells in the mouse, we examined tissue expression after transduced marrow transplantation of the drug selection gene, G156A mutant O6-methylguanine-DNA methyltransferase (G156A MGMT). To gain more experience with the rigor of the impact of selection on tissue-specific gene expression, we also asked whether there are expression differences between three different onco-retroviral backbones--MPSV, SF, and MFG. MGMT expression was compared after O6-benzylguanine (BG) and 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) drug selection in vivo. After mice were transplanted with cells transduced with MPSV, MFG, or SF retroviral vectors expressing G156A MGMT and drug treated, nearly complete replacement by transduced progenitors was observed in the marrow. Each backbone supported MGMT expression in all four hematopoietic lineages in vivo indicating that MGMT-mediated selection is indeed robust. Expression in marrow, spleen, and thymus was very similar between the vectors and differences were most likely due to differences in gene copy number per selected cell. In primary and secondary recipients, the highest expression was observed in MFG and this was the vector that transduced at the greatest proviral copy number per cell. These data indicate that strong selection pressure using the MGMT gene to protect primary and secondary repopulating murine stem cells from the toxicity of BCNU. Regardless of the vector backbone used, multiorgan expression was observed without evidence of gene silencing. These data help establish mutant, BG-resistant MGMT as a potent selection gene for stem cell selection in vivo.

Animals↗
PubMed2003

In vivo selection of MGMT(P140K) lentivirus-transduced human NOD/SCID repopulating cells without pretransplant irradiation conditioning.

Infusion of transduced hematopoietic stem cells into nonmyeloablated hosts results in ineffective in vivo levels of transduced cells. To increase the proportion of transduced cells in vivo, selection based on P140K O6-methylguanine-DNA-methyltransferase (MGMT[P140K]) gene transduction and O6-benzylguanine/1,3-bis(2-chloroethyl)-1-nitrosourea (BG/BCNU) treatment has been devised. In this study, we transduced human NOD/SCID repopulating cells (SRCs) with MGMT(P140K) using a lentiviral vector and infused them into BG/BCNU-conditioned NOD/SCID mice before rounds of BG/BCNU treatment as a model for in vivo selection. Engraftment was not observed until the second round of BG/BCNU treatment, at which time human cells emerged to compose up to 20% of the bone marrow. Furthermore, 99% of human CFCs derived from NOD/SCID mice were positive for provirus as measured by PCR, compared with 35% before transplant and 11% in untreated irradiation-preconditioned mice, demonstrating selection. Bone marrow showed BG-resistant O6-alkylguanine-DNA-alkyltransferase (AGT) activity, and CFUs were stained intensely for AGT protein, indicating high transgene expression. Real-time PCR estimates of the number of proviral insertions in individual CFUs ranged from 3 to 22. Selection resulted in expansion of one or more SRC clones containing similar numbers of proviral copies per mouse. To our knowledge, these results provide the first evidence of potent in vivo selection of MGMT(P140K) lentivirus-transduced human SRCs following BG/BCNU treatment.

Animals↗
PubMed2003

Pharmacokinetics of O6-benzylguanine (NSC637037) and its metabolite, 8-oxo-O6-benzylguanine.

O6-Benzylguanine and its metabolite, 8-oxo-O6-benzylguanine, are equally potent inhibitors of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase. Pharmacokinetic values are derived from cancer patients participating in a phase I trial (10 or 20 mg/m2 of O6-benzylguanine in a single bolus dose or 10 to 120 mg/m2 as a 60-min constant infusion). A two-compartment model fits the plasma concentration versus time profile of O6-benzylguanine. O6-Benzylguanine is eliminated rapidly from the plasma compartment in humans (t1/2 alpha and t1/2 beta are 2 +/- 2 min and 26 +/- 15 min [mean +/- SD, n = 7], respectively), and its plasma clearance (513 +/- 148 mL/min/m2) is not dose dependent. Metabolite kinetics are evaluated using both a novel approach describing the relationship between O6-benzylguanine and 8-oxo-O6-benzylguanine and classical metabolite kinetics methods. With increasing doses of O6-benzylguanine, the plasma clearance of 8-oxo-O6-benzylguanine, decreases, prolonging elimination of the metabolite. This effect is not altered by coadministration of BCNU. The urinary excretion of drug and metabolites is minimal.

Aged↗
PubMed2003

Blockage of abasic site repair enhances antitumor efficacy of 1,3-bis-(2-chloroethyl)-1-nitrosourea in colon tumor xenografts.

Methoxyamine (MX) has been shown to potentiate the antitumor effect of temozolomide (TMZ) in human tumor xenograft models. This potentiation is due to the reactivity of MX with apurinic/pyrimidinic (AP) sites in DNA, which are formed following DNA glycosylase removal of TMZ-induced methyl-purine adducts. MX-bound AP sites cannot be further processed by base excision repair (BER), resulting in cell death. On the basis of this finding, we investigated in vivo whether MX enhanced therapeutic efficacy of other agents, such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) that generates AP sites during DNA repair. Nude mice carrying human colon tumor xenografts, HCT116 and HCT116-Ch3, were treated by a single injection (i.p) of BCNU alone (30 mg/kg) or MX (2 mg/kg) combined with BCNU. The effect on tumor growth of BCNU alone was very moderate. Combined administration of MX and BCNU produced significant inhibition of tumor growth. Tumor growth delays were 14 +/- 3 days in HCT116 and 16 +/- 2 days in HCT116-Ch3 tumors, respectively (P < 0.05 versus control or BCNU alone groups). Similar results were also observed in SW480 and DLD1 tumors. Importantly, no systemic toxicity was noted with BCNU and MX. In contrast, BCNU (at dose of 25 mg/kg) combined with O(6)-benzylguanine (BG), an inhibitor of O(6)-alkylguanine-DNA alkyltransferase (AGT) being tested in clinical trials, caused toxic death in all treated mice. However, a lower dose BCNU (10 mg/kg) combined with BG and MX had significant antitumor effect without toxic death. Thus, targeting BER with MX is a promising strategy to improve the antitumor activity of BCNU and perhaps other DNA-damaging agents.

Animals↗
PubMed2002

Lung tumorigenesis associated with erb-B-2 and erb-B-3 overexpression in human erb-B-3 transgenic mice is enhanced by methylnitrosourea.

Erb-B-3 overexpression is associated with poor prognosis in non-small cell lung cancer and is often overexpressed in breast cancers. MMTVhuman-erb-B-3 transgenic mice were generated to evaluate the impact of erb-B-3 overexpression on lung and mammary gland tumorigenesis. These transgenic mice developed a high incidence of lung adenocarcinomas but not mammary gland tumors. The tumors overexpressed transgenic human [h]-erb-B-3 but also overexpressed endogenous erb-B-2, indicating that the heterodimer of h-erb-B-3-erb-B-2 was required for proliferative signal transduction to the nucleus. Lung tumor latency was shorter and the incidence higher in erb-B-3 transgenic mice treated with the methylating agent, methylnitrosourea [MNU]. In MNU treated mice, K-ras activating point mutations in codon 12, synergized with h-erb-B-3 in lung tumorogenesis. In bitransgenic MMTVrat-erb-B2/MMTV-human-erb-B-3 mice, lung tumor latency was also significantly shortened. Unlike over-expression of rat-erb-B-2, overexpression of h-erb-B-3 did not alter the incidence or latency of mammary tumors. Coupled erb-B-2 and erb-B-3 overexpression as well as K-ras activation induced signaling through mitogen-activated protein kinase (MAPK). This animal model links erb-B-3 with lung cancer, suggests that erb-B-2 and erb-B-3 heterodimerization is a necessary intermediate, and documents latency shortening by methylating agent-induced mutation of K-ras. This erb-B-3 mouse lung cancer model will help dissect genetic changes in lung tumorigenesis and may be useful for chemoprevention studies.

Animals↗
PubMed2002

Chemotherapy-induced O(6)-benzylguanine-resistant alkyltransferase mutations in mismatch-deficient colon cancer.

The ability of O(6)-benzylguanine (BG) to inactivate alkyltransferase (AGT) to potentiate the antitumor efficacy of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is being tested in clinical trials. As of now, there are no examples of acquired resistance to BG+BCNU in the clinical setting. However, we hypothesized that genetically unstable tumors might develop resistance to the combination after repeated drug-exposures to achieve therapeutic efficacy. To evaluate this possibility, we treated three colon cancer cell lines that are either proficient in mismatch repair (MMR) [SW480 (MMR wild type)] or deficient in MMR [HCT116 (hMLH1 mutant) and HCT15 (hMSH6 mutant)] with three cycles of BG+BCNU. After drug-treatments, HCT116 and HCT15 were completely resistant to BG-potentiated cytotoxicity of BCNU. In these two cell lines, the acquired BG resistance resulted from two de novo and different mutations at amino acid 165 in AGT: 165-lysine (K) to glutamic acid (E) (K165E in HCT116), and 165-lysine to asparagine (N) (K165N in HCT15). Both K165-mutated AGTs had markedly decreased enzymatic activity because of unstable AGT protein but were remarkably resistant to BG inactivation. FISH analysis showed that only one copy of MGMT gene exists in HCT116 cells, and the status of promoter methylation of MGMT in HCT15 showed that one allele of the MGMT promoter has an aberrant methylation. Thus, the MGMT gene expressing AGT either from one copy (HCT116) or from unmethylated allele (HCT15) was mutated because of the exposure to BG+BCNU in these two MMR-deficient cell lines. Conversely, MMR-proficient SW480 cells, treated with three cycles of BG+BCNU, maintained wt AGT and the sensitivity to BG-potentiated BCNU-cytotoxicity. To confirm that K165-mutated AGT proteins were responsible for resistance to BG+BCNU, we transfected K165E and K165N MGMT cDNAs into Chinese hampster ovary (CHO) cells. Transfected CHO cells had low AGT activity but increased IC(50) for either BCNU or temozolomide (TMZ), compared with parental CHO cells. BG did not potentiate the cytotoxicity of these two alkylating agents at concentrations up to 200 microM; in contrast, BG, at 25 microM, sensitized CHO-AGT (transfected with wt MGMT cDNA) cells to BCNU or TMZ-cytotoxicity by 3-4 fold. These results suggest that K165 AGT mutants arising in MMR-deficient tumor cells after treatment with chemotherapeutic agents are both resistant to BG-inactivation and are active in the repair of alkylated DNA adducts.

Adaptor Proteins, Signal Transducing↗
PubMed2002

Clinical relevance of MGMT in the treatment of cancer.

Anumber of DNA-damaging chemotherapeutic agents attack the O(6) position on guanine, forming the most potent cytotoxic DNA adducts known. The DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT), encoded by the gene MGMT, repairs alkylation at this site and is responsible for protecting both tumor and normal cells from these agents. Cells and tissues vary greatly in AGT expression, not only between tissues but also between individuals. AGT activity correlates inversely with sensitivity to agents that form O(6)-alkylguanine DNA adducts, such as carmustine (BCNU), temozolomide, streptozotocin, and dacarbazine. The one exception is those tumors lacking mismatch repair, which renders them resistant to methylating agents. A recent study in patients with gliomas confirmed the correlation between low-level expression of the MGMT gene and response and survival after BCNU. An inhibitor to AGT, O(6)-benzylguanine (BG), depletes AGT in human tumors without associated toxicity and is now in phase II clinical trials. Finally, mutations within the active site region of the MGMT gene render the AGT protein resistant to BG inactivation. As a result, mutant MGMT gene transfer into hematopoietic stem cells has been shown to selectively protect the marrow from the combination of an alkylating agent and BG, while at the same time sensitizing tumor cells. MGMT remains a paradigm for development of new agents that modulate known mechanisms of drug resistance in cancer cells and raise the spectra of combinatorial therapies that encompass known drug resistance mechanisms.

Antineoplastic Agents↗
PubMed2002

Adult bone marrow stem cells for cell and gene therapies: implications for greater use.

There is excitement generated almost daily about the possible uses of stem cells to treat human disease. Much of the interest of late is generated by embryonic stem cells (ESCs). As exciting as ESCs may be, they are quite controversial for moral reasons, given their source. They are also scientifically controversial since they are much less well understood than the original, long-standing, and clinically successful hematopoietic stem cell (HSC). HSCs have the distinct advantage of being reasonably well characterized and have been proven in the clinic. They can be isolated by simple procedures directly from the bone marrow or from peripheral blood after being stimulated (mobilized). They can then be manipulated and delivered to a patient, often producing a cure. Their biology provides the paradigm by which all other stem cells are judged, and they have little in the way of moral controversy surrounding them given they are isolated from adults who have consented to the procedure. Another putative stem cell has gained momentum in the last few years; the mesenchymal stem cell (MSC). MSCs appear to have much in common with HSCs. They were originally characterized from bone marrow, are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. Unlike HSCs, they have not yet been definitively shown to function as stem cells, despite their ability to differentiate into various mesenchymal cell types under the right culture conditions. Still, there is mounting evidence these cells may be useful, if not as true stem cells then at least as vehicles for emerging cell and gene therapies, especially in the field of tissue engineering. While this is an important endpoint, it is more important to thoroughly understand stem cell biology. That understanding can then be applied toward the ultimate goal of using these cells not just for various forms of therapy, but rather as a tool to discover the mechanisms and means to bring about directed repair and regeneration of damaged or diseased tissues and organs. The excitement of HSCs and MSCs has been muted somewhat by the excitement surrounding ESCs, primarily due to the fact HSCs and MSCs are viewed as limited to specific cell types while ESCs could potentially be applied to any cell type. Recent information indicates HSCs, MSCs, and other cells in general may have more universal differentiation abilities than previously thought.

Animals↗
PubMed2002

Lentiviral transduction of P140K MGMT into human CD34(+) hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro.

Lentiviral vectors may improve hematopoietic stem cell (HSC) gene transfer because of their enhanced ability to transduce nondividing cells. However, many studies report efficient transduction only at high multiplicities of infection (MOI). This study reports efficient transduction of human CD34(+) cells with a drug resistance gene allowing post-transduction selection using lentivirus under low-MOI conditions that did not require cytokine stimulation or viral concentration. We used the P140K methylguanine-DNA-methyltransferase mutant (P140K MGMT) as the gene insert into a second-generation lentiviral backbone and triple-plasmid transfection to generate vesicular stomatitis virus (VSV)-G protein-pseudotyped virus. The P140K MGMT gene product, O(6)-alkylguanine-DNA-alkyltransferase (AGT), provides protection from the therapeutic drug combination of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the wild-type AGT inhibitor O(6)-benzylguanine (BG). Low-speed spinoculation enhanced transduction more than addition of Polybrene or multiple virus exposures. Addition of cytokines was not required. Low-MOI transduction (< or =1) of human CD34(+) and CD34(+) lin(-) cells with P140K MGMT lentivirus resulted in an average 41% and 89% gene transfer rate as assessed by PCR, respectively, and concordant AGT expression that conferred substantial clonogenic survival advantage after BG/BCNU treatment. During in vitro drug selection, 87% of surviving CD34(+) cell-derived colony-forming units (CFU) were transduced. This work shows the potential utility of lentiviral vectors for drug resistance gene transfer to HSCs for the purpose of in vivo selection and marrow protection. Because drug selection will enrich for transduced progenitors, high MOI can be avoided, improving the safety profile of lentiviral gene transfer.

Antigens, CD34↗
PubMed2002

Hematologic effects of linezolid: summary of clinical experience.

Linezolid has been associated with reversible myelosuppression. Clinical trial data were evaluated for anemia, thrombocytopenia, and neutropenia. Thrombocytopenia and a slight increased risk for anemia were evident at > or =2 weeks of linezolid treatment. Hematologic abnormalities were consistent with mild, reversible, duration-dependent myelosuppression. Appropriate monitoring is warranted with linezolid use.

Acetamides↗
PubMed2002

Cancer gene therapy: scientific basis.

Gene therapy of cancer has been one of the most exciting and elusive areas of therapeutic research in the past decade. Critical developments have occurred in gene therapy targeting cancer cells, cancer vasculature, the immune system, and the bone marrow, itself often the target for severe toxicity from therapeutic agents. We review some recent developments in the field. In each instance, clear preclinical models validated the therapeutic approach and efforts have been made to evaluate the target impact in both preclinical and early clinical trials. Although no cures can consistently be expected from today's cancer gene therapy, the rapid progress may imply that such cures are a few short years away.

Animals↗