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T L Kindwall-Keller,
Y Hegerfeldt,
H J Meyerson,
S Margevicius,
P Fu,
W van Heeckeren,
H M Lazarus,
B W Cooper, S L Gerson,
P Barr,
W W Tse,
C Curtis,
L R Fanning,
R J Creger,
J M Carlson-Barko,
M J Laughlin
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ABSTRACT: As the threshold nucleated cell dose for one-unit umbilical cord blood (UCB) in adults has not to date been firmly established, we prospectively compared one- vs two-unit UCB transplantation after reduced intensity conditioning (RIC) in adult patients with hematological malignancies. Study design specified one-UCB unit if the cryopreserved total nucleated cell (TNC) dose was 2.5 × 10(7)/kg recipient weight, otherwise two units matched at minima of 4/6 HLA loci to the patient and 3/6 to each other were infused. A total of 27 patients received one unit; 23 patients received two units. Median time to ANC >500/μL was 24 days (95% confidence interval 22-28 days), 25 days for one unit and 23 days for two units (P=0.99). At day 100, ANC >500/μL was 88.4 and 91.3% in the one- and two-unit groups (P=0.99), respectively. Three-year EFS was 28.6% and 39.1% in the one- and two-unit groups (P=0.71), respectively. Infusion of two units was associated with a significantly lower relapse risk, 30.4% vs 59.3% (P=0.045). Infused cell doses (TNC, CD3(+), CD34(+) and CD56(+)CD3(neg)) did not impact on engraftment, OS or EFS. Taken together, one-unit UCB transplantation with a threshold cell dose 2.5 × 10(7)/kg recipient weight after RIC is a viable option for adults, although infusion of two units confers a lower relapse incidence.
Bone marrow transplantation 10/2011; 47(7):924-33. · 3.00 Impact Factor
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Hillard M Lazarus,
Scott R Sommers,
Lisa M Arfons,
Pingfu Fu,
S A Ataergin,
N M Kaye,
F Liu,
Tamila L Kindwall-Keller,
Brenda W Cooper,
Mary J Laughlin,
Richard J Creger,
Paul M Barr, Stanton L Gerson,
David Kaplan
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ABSTRACT: Nine plasma cell myeloma patients spontaneously developed histologically proven autologous graft-versus-host disease (GVHD) limited predominantly to the gastrointestinal tract within 1 month of initial autologous hematopoietic cell transplantation (AHCT) using high-dose melphalan conditioning. All recipients responded promptly to systemic and nonabsorbable oral corticosteroid therapy. All patients previously received systemic therapy with thalidomide, lenalidomide, or bortezomib before AHCT. Using enzymatic amplification staining-enhanced flow cytometry, we evaluated expression of selected transcription regulators, pathway molecules, and surface receptors on samples of the infused hematopoietic cell grafts. We demonstrated significantly enhanced expression of GATA-2, CD130, and CXCR4 on CD34(+) hematopoietic progenitor cells of affected patients compared with 42 unaffected AHCT controls. These 3 overexpressed markers have not been previously implicated in autologous GVHD. Although we did not specifically evaluate T cells, we postulate that exposure over time to the various immunomodulating therapies used for induction treatment affected not only the CD34(+) cells but also T cells or relevant T cell subpopulations capable of mediating GVHD. After infusion, the affected hematopoietic progenitor cells then encounter a host that has been further altered by the high-dose melphalan preparative regimen; such a situation leads to the syndrome. These surface markers could be used to develop a model to predict development of this syndrome. Autologous GVHD potentially is a serious complication of AHCT and should be considered in plasma cell myeloma patients with otherwise unexplained gastrointestinal symptoms in the immediate post-AHCT period. Prompt recognition of this condition and protracted treatment with nonabsorbable or systemic corticosteroids or the combination may lead to resolution.
Biology of blood and marrow transplantation: journal of the American Society for Blood and Marrow Transplantation 03/2011; 17(7):970-8. · 3.15 Impact Factor
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P Fu,
W J van Heeckeren,
P D Wadhwa,
D J Bajor,
R J Creger,
Z Xu,
B W Cooper,
M J Laughlin, S L Gerson,
O N Koç,
H M Lazarus
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ABSTRACT: Evaluation of time to event outcomes usually is examined by the Kaplan-Meier method and Cox proportional hazards models. We developed a modified statistical model based on histologic grade and other variables to describe the time-dependent outcome for autologous stem cell transplant (autotransplant) performed for non-Hodgkin's lymphoma (NHL) based on histologic grade and other variables. One hundred and fourteen relapsed or refractory NHL patients were treated using BCNU 600 mg/m2, etoposide 2400 mg/m2, and cisplatin 200 mg/m2 IV followed by autotransplant. Median age was 53.5 (range: 25-70) years, 78 patients had aggressive NHL and 36 indolent NHL. Seventy-five patients received involved-field radiotherapy just prior to transplant. At a median follow-up of 33 (range: 3 to 118) months, the estimated 5-year Kaplan-Meier probabilities of overall survival and disease-free survival were 61% and 51%, respectively. Cox proportional hazards model analysis showed that proportionality did not hold for lymphoma grade, indicating that the relationship between the grade and disease-free survival differed over time. By piece-wise Cox model, the relative risk for experiencing relapse or death after 1 year in patients with indolent compared with patients with aggressive NHL was 2.81 (p=0.019) with 95% confidence interval (1.19, 6.65). The time-dependent effect of lymphoma grade on disease-free survival suggests the need for early (within first year) incorporation of novel therapeutic approaches in management of patients with indolent NHL undergoing autotransplant.
Contemporary Clinical Trials 04/2008; 29(2):157-64. · 1.81 Impact Factor
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P Fu,
R K Bagai,
H Meyerson,
D Kane,
R M Fox,
R J Creger,
B W Cooper, S L Gerson,
M J Laughlin,
O N Koc,
H M Lazarus
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ABSTRACT: We examined pre-mobilization blood CD34+ cell count to predict ability to mobilize adequate peripheral blood progenitor cells (PBPC) in 106 cancer patients and 36 allogeneic donors. Mean pre-mobilization therapy blood CD34+ cell count was 3.1 cells x 10(6)/l (s.d. = 3.9, r = 0.3-37) and mean CD34+ cells collected were 5.3 x 10(6) cells/kg/leukapheresis procedure (s.d. = 7.0, r = 0.03-53). Yields correlated with pre-mobilization CD34+ cells x 10(6)/l (r = 0.37, P-value < 0.0001); correlation was stronger in allogeneic donors (r = 0.56, P-value = 0.0004) and males (r = 0.46, P-value < 0.0001). Based on classification and regression tree multivariate analysis, the predictive value of pre-mobilization blood CD34+ cell count was confounded by other variables, including age, gender, mobilization regimen and malignancy type. We generated an algorithm to predict a minimum PBPC yield of 1 x 10(6) CD34+ cells/kg/leukapheresis procedure after mobilization. A threshold pre-mobilization blood CD34+ cell count of 2.65 cells x 10(6)/l was the most important decision point in predicting successful mobilization. Only 2% of subjects with pre-mobilization blood CD34+ cell counts > 2.65 cells x 10(6)/l did not achieve the minimum per apheresis, whereas 24% with pre-mobilization values below threshold had inadequate mobilization. Prospectively identifying individuals at risk for mobilization failure would allow for improved treatment planning, resource utilization and time saving.
Bone Marrow Transplantation 08/2006; 38(3):189-96. · 3.75 Impact Factor
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ABSTRACT: Genomic stability is essential for cell and organism longevity. Without genomic stability, replication errors and external stress as well as direct forms of DNA damage can induce mutations, which decrease cell survival, cause altered gene expression, and can lead to cellular transformation. All represent the antithesis of maintenance of normal stem cell function. We argue here that genomic stability is essential for stem cell maintenance and longevity. This concept is supported by human diseases associated with premature aging and animal models of DNA damage repair abnormalities all of which lead to abnormalities of stem cell survival. Furthermore, with competitive repopulation, hematopoietic stem cell survival can be assessed in the face of DNA repair defects, and results from these studies support the general conclusion that chemotherapy and other forms of DNA damage lead to stem cell failure syndromes and malignant transformation most commonly along the myeloid and lymphoid pathways. Thus one origin of the cancer stem cell phenotype is the inability to maintain genomic stability among the stem cell population leading to mutational alterations and transformation. Capturing stem cells at this transition point represents an exciting field of discovery possibly leading to early detection and therapeutic interventions.
Ernst Schering Foundation symposium proceedings. 02/2006;
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ABSTRACT: The concept of hematopoietic stem cell gene therapy is as exciting as that of stem cell transplantation itself. The past 20 years of research have led to improved techniques for transferring and expressing genes in hematopoietic stem cells and preclinical models now routinely indicate the ease with which new genes can be expressed in repopulating stem cells of multiple species. Both modified murine oncoretroviruses and lentiviruses transmit genes into the genome of hematopoietic stem cells and allow expression in the host following transplantation. Using oncoretroviruses, therapeutic genes for severe combined immunodeficiency, common variable gamma chain immunodeficiency, chronic granulomatous disease, Hurler's and Gaucher's Disease have all been used clinically with only modest success except for the patients with immunodeficiency in whom a partial T-cell chimerism has been dramatic. Since stem cell selection in vivo appears important to the therapeutic success of gene transfer, drug resistance selection, most recently using the MGMT gene, has been developed and appears to be safe. Future trials combining a drug resistance and therapeutic gene are planned, as are trials using safety-modified lentiviruses. The therapeutic potential of hematopoietic stem cell gene therapy, particularly given recent advances in stem cell plasticity, remains an exceptionally exciting area of clinical research.
Bone Marrow Transplantation 08/2003; 32(1):1-7. · 3.75 Impact Factor
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ABSTRACT: Patients with Hurler syndrome (mucopolysaccharidosis type-IH) and metachromatic leukodystrophy (MLD) develop significant skeletal and neurologic defects that limit their survival. Transplantation of allogeneic hematopoietic stem cells results in partial correction of the clinical manifestations. We postulated that some of these defects may be corrected by infusion of allogeneic, multipotential, bone marrow-derived mesenchymal stem cells (MSC). Patients with Hurler syndrome (n = 5) or MLD (n = 6) who previously underwent successful bone marrow transplantation from an HLA-identical sibling were infused with 2-10 x 10(6)/kg MSCs, isolated and expanded from a bone marrow aspirate of the original donor. There was no infusion-related toxicity. In most recipients culture-purified MSCs at 2 days, 30-60 days and 6-24 months after MSC infusion remained of host type. In two patients the bone marrow-derived MSCs contained 0.4 and 2% donor MSCs by FISH 60 days after MSC infusion. In four patients with MLD there were significant improvements in nerve conduction velocities after MSC infusion. The bone mineral density was either maintained or slightly improved in all patients. There was no clinically apparent change in patients' overall health, mental and physical development after MSC infusion. We conclude that donor allogeneic MSC infusion is safe and may be associated with reversal of disease pathophysiology in some tissues. The role of MSCs in the management of Hurler syndrome and MLD should be further evaluated.
Bone Marrow Transplantation 09/2002; 30(4):215-22. · 3.75 Impact Factor
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ABSTRACT: Human bone marrow-derived mesenchymal stem cells (hMSCs) are being investigated for a potential therapeutic role as hematopoietic support cells following chemo-radiotherapy and as vehicles of gene delivery. Although hMSCs can be safely infused into humans and experimental animals, there is limited evidence regarding their engraftment and proliferation in vivo. We developed a drug resistance gene transfer strategy to mark and selectively enrich marked hMSCs using chemotherapy. We have determined that hMSCs are markedly sensitized to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in vitro when pretreated with O(6)-benzylguanine (BG) resulting in a more than four-fold decrease in BCNU IC(90). The MFG retroviral vector encoding a bicistronic transcript for green fluorescent protein (GFP) and mutant (G156A)-methylguanine methyltransferase (G156A-MGMT), which encodes O(6)-alkylguanine-DNA alkyltransferase (AGT), conferring, BG plus BCNU resistance, transduced a high percentage of hMSCs. Transduced hMSCs had high expression of GFP and AGT and became significantly resistant to BG and BCNU. Furthermore, the proportion of GFP expressing transduced hMSCs increased from 32 +/- 14% to 70 +/- 14% following BG and BCNU treatment in vitro. Intravenously infused hMSCs were detected in NOD-SCID mice 8 weeks later by PCR analysis but could not be recultured from the bone marrow. GFP-expressing hMSCs inoculated into subcutaneous wounds in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mouse could be recultured at a low frequency, but enriched by BG and BCNU treatment from 0.05 +/- 0.03% to 0.55 +/- 0.4 (p = 0.028, Welch t-test). Our results indicate that hMSCs are sensitive to BG and BCNU, predicting significant toxicity to the hematopoietic microenvironment with this therapy. G156A-MGMT is a powerful selectable gene for a second marker gene in hMSCs. Drug resistance gene transfer into hMSCs may allow in vivo enrichment of hMSCs when MSC homing and engraftment into target tissues is optimized.
Journal of Hematotherapy & Stem Cell Research 11/2001; 10(5):691-701.
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ABSTRACT: In the setting of target-based anticancer drug development, it is critical to establish that the observed preclinical activity can be attributed to modulation of the intended target in early phase trials in human subjects. This paradigm of target modulation allows us to determine a Phase II or III dose (optimal biochemical/biological modulatory dose) that may not necessarily be the maximum tolerated dose. A major obstacle to target-based (often cytostatic) drug development has been obtaining relevant tumor tissue during clinical trials of these novel agents for laboratory analysis of the putative marker of drug effect.
From 1989 to present, we have completed seven clinical trials in which the end point was a biochemical or biological modulatory dose in human tumor tissues (not surrogate tissue). Eligibility enrollment required that patients have a biopsiable lesion either with computerized tomography (CT) guidance or direct visualization and consent to sequential (pre and posttreatment) biopsies.
A total of 192 biopsies were performed in 107 patients. All but 8 patients had sequential pre and posttreatment biopsies. Seventy-eight (73%) of the 107 patients had liver lesion biopsies. In eight patients, either one or both biopsies contained insufficient viable tumor tissue or no tumor tissue at all for analysis. Of a total of 99 patients in whom we attempted to obtain paired biopsies, a total of 87 (88%) were successful. Reasons for failure included patient refusal for a second biopsy (n = 2), vasovagal reaction with first biopsy precluding a second biopsy (n = 1), subcapsular hepatic bleeding (n = 1), and most commonly obtaining necrotic tumor, fibrous, or normal tissue in one of the two sequential biopsies (n = 8).
This is the first and largest reported series demonstrating that with adequate precautions and experience, sequential tumor biopsies are feasible and safe during early phase clinical trials.
Clinical Cancer Research 11/2001; 7(10):2971-6. · 7.74 Impact Factor
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ABSTRACT: The erbB family of receptor tyrosine kinases is frequently implicated in neoplasia. Amplification and overexpression of erbB2/neu has been found in 20 to 40% of human breast cancers. Previous studies using MMTV/c-neu transgenic mice have linked rat neu overexpression to mammary tumor development. In this study, we provide evidence that rat neu overexpression in mammary tumors of MMTV/c-neu transgenic mice is always associated with demethylation of the MMTV promoter, whereas the normal mammary glands of these transgenic mice always contain specific methylated regions of the MMTV promoter. In addition, after exposure to N-methyl-N-nitrosourea (MNU), the latency of mammary tumor development is significantly reduced and again is also associated with MMTV promoter demethylation. Thus, the transition from methylation to hypomethylation of the MMTV promoter induces high-level expression of c-neu and appears to be a prerequisite for transformation from normal to malignant mammary epithelium, either spontaneously or after carcinogen exposure. Expression of transgenic c-neu from the demethylated MMTV promoter appears to be an early event that allows outgrowth of mammary epithelium predisposed to malignant transformation.
Oncogene 10/2001; 20(42):6009-17. · 6.37 Impact Factor
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ABSTRACT: O6-alkylguanine DNA alkyltransferase (AGT) is a key mechanism in the prevention against MNU induced malignant transformation by removal of O6 methyl guanine (O6mG) adducts. We asked whether heterozygous p53 deficient mice (p53+/-) would be more susceptible to MNU induced lymphomas than wild type mice, and whether O6mG adducts were responsible for this susceptibility. To determine whether MGMT overexpression would be protective, p53+/- mice were bred to human MGMT transgenic mice (MGMT+) and treated with 50 mg/kg MNU. MNU increased the incidence of thymic lymphomas in non-transgenic p53+/- mice from 23% (n=13) to 68% (n=22) and decreased the mean latency from 433 to 106 days (P=0.01 compared to untreated mice). Wild type mice had an incidence of 30% (n=38) and a mean latency of 135 days after MNU. Overexpression of MGMT in the thymus of p53+/- mice significantly reduced the lymphoma incidence from 68 to 28% (n=17) and increased the latency from 106 to 167 days (P=0.003). Similarly, the lymphoma incidence in MGMT+/wild type mice decreased from 30 to 8% (n=12) and the latency increased to 297 days (P=0.2). Loss of the wild type allele was found in only 2/17 lymphomas occurring in p53+/- mice and there were no significant point mutations in exons 5-8 of p53. Furthermore, there was no loss of p53 function in these mice. These data demonstrate that unrepaired O6mG lesions act cooperatively with the reduced p53 dose and lead to lymphomagenesis in p53+/- mice, but AGT overexpression and rapid removal of O6mG adducts is protective.
Oncogene 09/2001; 20(38):5258-63. · 6.37 Impact Factor
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ABSTRACT: Temozolomide (TMZ) is a methylating agent of the imidotetrazine class, whose cytotoxic product is O(6)-methylguanine DNA adducts, which initiate a futile recycling of the mismatch repair pathway causing DNA strand breaks and apoptotic cell death in mismatch repair proficient cells. The DNA repair protein O(6)-alkylguanine DNA alkyltransferase (AGT) repairs these adducts in a suicide manner and reduces the cytotoxic action of TMZ. An antitumor threshold is reached when sufficient adducts are formed by TMZ to inactivate AGT. In this study, we evaluated the relation between TMZ dosing and AGT depletion in patients with deep visceral tumors and in peripheral blood mononuclear cells (PBMCs) to determine whether the dose of TMZ was sufficient to inactivate AGT and lead to therapeutic efficacy. To do so, we compared single dose therapy with a novel twice daily regimen in a laboratory correlate-driven Phase I dose escalation study. p.o. bolus dose TMZ 200 mg/m(2) daily times five was compared with the same bolus on day 1 followed by nine doses at 12-h intervals of 50, 75, 90, or 100 mg/m(2). Dose-limiting toxicity in the bid regimen (grade IV thrombocytopenia and neutropenia) was seen at 100 mg/m(2), cumulative dose 1100 mg/m(2), and the maximum tolerated dose was 1010 mg/m(2). The degree of tumor tissue AGT activity depletion measured in biopsies before and on day 5 of therapy varied widely, between 0 (in 3 patients) and 99% (in 1), with the majority of patients (10 of 15) having 52-84% tumor AGT depletion. In contrast, AGT activity in PBMCs fell rapidly during TMZ administration to undetectable levels in all dosage groups on day 5 but did not correlate with tumor AGT depletion. TMZ pharmacokinetics were dose proportional; no accumulation occurred >5-day period in the bid regimen. Two partial responses were seen, lasting 3 and 4 months. Five additional patients achieved prolonged stabilization of disease for 4-6 monthly cycles. This is the first study to document that at maximum tolerated doses, TMZ depletes PBMC AGT but only partially and variably depletes visceral tumor AGT in most patients, even during twice daily dosing. Drug combinations or schedules designed to maximally deplete tumor AGT might improve TMZ efficacy.
Clinical Cancer Research 08/2001; 7(8):2309-17. · 7.74 Impact Factor
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ABSTRACT: O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs O(6)-alkylating DNA adducts generated by alkylating therapeutic agents. Therefore, AGT activity may be an important marker of tumor and normal tissue sensitivity to chemotherapeutic agents and a predictor for the success of chemotherapeutic regimens. It is rapidly inactivated by O(6)-benzylguanine (BG) that mimics its substrates, O(6)-methylguanine and O(6)-chloroethylguanine DNA adducts. In a Phase I clinical trial, BG was given in increasing doses (from 10 to 120 mg/m(2)) by 1-h infusion. We previously reported depletion of AGT activity, and in this report, we demonstrate the relationship between degradation of BG-inactivated AGT protein and the depletion of AGT activity in peripheral blood mononuclear cells (PBMCs) and tumor samples obtained by computed tomography-guided cutting needle biopsy from patients prior to BG and either 2 or 18 h after BG. In PBMCs, BG inactivated AGT activity by over 95-100% at the end of a 1-h infusion, and depletion was maintained for 18 h. In contrast, AGT protein remained almost unchanged for up to 18 h after BG, suggesting that inactivated AGT proteins remain immunoreactive and are not rapidly degraded in PBMCs. In patient tumor biopsies, AGT activity was depleted approximately 90% 2 h after BG. Tumor AGT protein levels were reduced to approximately 40% of pretreatment values when detected by either Western blot or immunohistochemistry staining. In tumor samples obtained 18 h after BG, >95% inactivation of tumor AGT activity was observed at BG doses of 36-80 mg/m(2), and complete depletion of tumor AGT activity occurred at 120 mg/m(2) BG. However, residual AGT protein (5-10% of baseline) was detectable in all tumor samples. Therefore, the degradation of BG-inactivated AGT protein appeared to be much more rapid in tumors than that in PBMCs, which may impact on AGT regeneration rates as well. Because degradation of BG-inactivated AGT takes place slowly, antibody-based measurements of AGT protein correlate poorly with depletion of AGT activity immediately after BG. Thus, biochemical activity measurements remain the appropriate monitor of AGT during therapeutic modulation. These data provide the first and conclusive evidence of differential degradation rates of inactivated AGT in PBMCs and tumors of patients after treatment with BG and suggest that immunoreactive AGT measurements in PBMCs are a poor surrogate for AGT activity in tumor tissue.
Clinical Cancer Research 08/2001; 7(8):2318-24. · 7.74 Impact Factor
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M J Laughlin,
J Barker,
B Bambach,
O N Koc,
D A Rizzieri,
J E Wagner, S L Gerson,
H M Lazarus,
M Cairo,
C E Stevens,
P Rubinstein,
J Kurtzberg
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ABSTRACT: Umbilical-cord blood from unrelated donors who are not HLA-identical with the recipients can restore hematopoiesis after myeloablative therapy in children. We studied the use of transplantation of umbilical-cord blood to restore hematopoiesis in adults.
Sixty-eight adults with life-threatening hematologic disorders received intensive chemotherapy or total-body irradiation and then transplants of HLA-mismatched umbilical-cord blood. We evaluated the outcomes in terms of hematologic reconstitution, the occurrence of acute and chronic graft-versus-host disease (GVHD), relapses, and event-free survival.
Of the 68 patients, 48 (71 percent) received grafts of umbilical-cord blood that were mismatched for two or more HLA antigens. Of the 60 patients who survived 28 days or more after transplantation, 55 had neutrophil engraftment at a median of 27 days (range, 13 to 59). The estimated probability of neutrophil recovery in the 68 patients was 0.90 (95 percent confidence interval, 0.85 to 1.0). The presence of a relatively high number of nucleated cells in the umbilical-cord blood before it was frozen was associated with faster recovery of neutrophils. Severe acute GVHD (of grade III or IV) occurred in 11 of 55 patients who could be evaluated within the first 100 days after transplantation. Chronic GVHD developed in 12 of 33 patients who survived for more than 100 days after transplantation. The median follow-up for survivors was 22 months (range, 11 to 51). Of the 68 patients, 19 were alive and 18 of these (26 percent) were disease-free 40 months after transplantation. The presence of a high number of CD34+ cells in the graft was associated with improved event-free survival (P=0.05).
Umbilical-cord blood from unrelated donors can restore hematopoiesis in adults who receive myeloablative therapy and is associated with acceptable rates of severe acute and chronic GVHD.
New England Journal of Medicine 07/2001; 344(24):1815-22. · 53.30 Impact Factor
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ABSTRACT: We have previously shown that human cancer cells deficient in DNA mismatch repair (MMR) are resistant to the chemotherapeutic methylating agent temozolomide (TMZ) and can be sensitized by the base excision repair (BER) blocking agent methoxyamine (MX) [21]. To further characterize BER-mediated repair responses to methylating agent-induced DNA damage, we have now evaluated the effect of MX on TMZ-induced DNA single strand breaks (SSB) by alkaline elution and DNA double strand breaks (DSB) by pulsed field gel electrophoresis in SW480 (O6-alkylguanine-DNA-alkyltransferase [AGT]+, MMR wild type) and HCT116 (AGT+, MMR deficient) colon cancer cells. SSB were evident in both cell lines after a 2-h exposure to equitoxic doses of temozolomide. MX significantly increased the number of TMZ-induced DNA-SSB in both cell lines. In contrast to SSB, TMZ-induced DNA-DSB were dependent on MMR status and were time-dependent. Levels of 50 kb double stranded DNA fragments in MMR proficient cells were increased after TMZ alone or in combination with O6-benzylguanine or MX, whereas, in MMR deficient HCT116 cells, only TMZ plus MX produced significant levels of DNA-DSB. Levels of AP endonuclease, XRCC1 and polymerase beta were present in both cell lines and were not significantly altered after MX and TMZ. However, cleavage of a 30-mer double strand substrate by SW480 and HCT116 crude cell extracts was inhibited by MX plus TMZ. Thus, MX potentiation of TMZ cytotoxicity may be explained by the persistence of apurinic/apyrimidinic (AP) sites not further processed due to the presence of MX. Furthermore, in MMR-deficient, TMZ-resistant HCT116 colon cancer cells, MX potentiates TMZ cytotoxicity through formation of large DS-DNA fragmentation and subsequent apoptotic signalling.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2001; 485(4):269-81. · 2.85 Impact Factor
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ABSTRACT: Applied molecular evolution is a rapidly developing technology that can be used to create and identify novel enzymes that nature has not selected. An important application of this technology is the creation of highly drug-resistant enzymes for cancer gene therapy. Seventeen O(6)-alkylguanine-DNA alkyltransferase (AGT) mutants highly resistant to O(6)-benzylguanine (BG) were identified previously by screening 8 million variants, using genetic complementation in Escherichia coli. To examine the potential of these mutants for use in humans, the sublibrary of AGT clones was introduced to human hematopoietic cells and stringently selected for resistance to killing by the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. This competitive analysis between the mutants in human cells revealed three AGT mutants that conferred remarkable resistance to the combination of BG and 1,3-bis(2-chloroethyl)-1-nitrosourea. Of these, one was recovered significantly more frequently than the others. Upon further analysis, this mutant displayed a level of BG resistance in human hematopoietic cells greater than that of any previously reported mutant.
Proceedings of the National Academy of Sciences 05/2001; 98(9):4950-4. · 9.68 Impact Factor
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ABSTRACT: Rebeccamycin analog (NSC 655649) is active against a variety of both solid and nonsolid tumor cell lines. We performed a phase I trial to determine the maximum-tolerated dose (MTD) of rebeccamycin analog when given on a daily x 5 schedule repeated every 3 weeks, characterize the toxicity profile using this schedule, observe patients for antitumor response, and determine the pharmacokinetics of the agent and pharmacodynamic interactions.
Thirty assessable patients received a total of 153 cycles according to the following dose escalation schema: 60, 80, 106, 141, and 188 mg/m(2)/d x 5 days.
Grade 2 phlebitis occurred in all patients before the use of central venous access, placed at dose level 4 and higher. Dose-limiting toxicity (DLT), grade 4 neutropenia, occurred at 188 mg/m(2)/d x 5 days in both previously treated and chemotherapy-naive patients. Pharmacokinetic analysis revealed a three-compartmental model of drug elimination and a long terminal half-life (154 +/- 55 hours). The percentage drop in absolute neutrophil count correlates with the area under the curve infinity. The presence of a second peak during the elimination phase as well as a high concentration of NSC 655649 in biliary fluid compared with the corresponding plasma measurement (one patient) is suggestive of enterohepatic circulation. Two partial responses, two minor responses, and six prolonged (> 6 months) cases of stable disease were observed. Of these, three patients with gallbladder cancer and one patient with cholangiocarcinoma experienced either a minor response or a significant period of freedom from progression.
The recommended phase II dose for NSC 665649 on a daily x 5 every 3 weeks schedule is 141 and 165 mg/m(2)/d for patients with prior and no prior therapy, respectively, with DLT being neutropenia. During this phase I trial, encouraging antitumor activity was been observed.
Journal of Clinical Oncology 04/2001; 19(8):2309-18. · 18.37 Impact Factor
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ABSTRACT: Myelosuppression is commonly observed after alkylating agent chemotherapy due to low levels of O(6)-alkylguanine DNA alkyltransferase protein (AGT) in hematopoietic progenitors. Mice that lack AGT in all organs, O(6)-methylguanine-DNA methyltransferase gene knockout (MGMT(-/-)) mice are extremely hypersensitive to the methylating agent N-methyl-N-nitrosourea (MNU) and exhibit a 10-fold reduction in the LD(90). To determine whether bone marrow damage was the cause of the increased lethality, we transplanted 1 x 10(6) wild-type marrow into MGMT(-/-) mice and MGMT(-/-) marrow into wild-type mice and observed survival after MNU. Lethally irradiated MGMT(-/-) mice given > or = 25 mg/kg MNU 3 weeks after transplant of wild-type cells survived > 30 days (n = 11), whereas this dose was lethal to control MGMT(-/-) mice 9-12 days post treatment (n = 5). Conversely, lethally irradiated wild-type mice transplanted with MGMT(-/-) cells died after only 20-60 mg/kg MNU within 8-12 days (n = 6). No significant toxicities were found in other organs. Additionally, in an in vivo post transplant competition model, wild-type long-term repopulating cells had a > 200-fold competitive survival advantage over MGMT(-/-) cells, and after MNU treatment completely repopulated the mouse when transplanted at only one-tenth the cell number. We also observed a strong selection for transplanted marrow-derived wild-type stromal elements in the MGMT(-/-) background after drug treatment. These data indicate that alkylating agent hypersensitivity of MGMT(-/-) mice results from hematopoietic damage at the stem level. Thus, DNA repair involving AGT in hematopoietic cells is required for normal host survival following exposure to methylating and chloroethylating agents.
Journal of Hematotherapy & Stem Cell Research 03/2001; 10(1):115-23.
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S L Gerson
Experimental Hematology 01/2001; 28(12):1315-24. · 2.90 Impact Factor
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ABSTRACT: 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.
Journal of Hematotherapy & Stem Cell Research 01/2001; 9(6):841-8.
