Uracil DNA glycosylase (UNG) loss enhances DNA double strand break formation in human cancer cells exposed to pemetrexed.

Misincorporation of genomic uracil and formation of DNA double strand breaks (DSBs) are known consequences of exposure to TS inhibitors such as pemetrexed. Uracil DNA glycosylase (UNG) catalyzes the excision of uracil from DNA and initiates DNA base excision repair (BER). To better define the relationship between UNG activity and pemetrexed anticancer activity, we have investigated DNA damage, DSB formation, DSB repair capacity, and replication fork stability in UNG(+/+) and UNG(-/-) cells. We report that despite identical growth rates and DSB repair capacities, UNG(-/-) cells accumulated significantly greater uracil and DSBs compared with UNG(+/+) cells when exposed to pemetrexed. ChIP-seq analysis of γ-H2AX enrichment confirmed fewer DSBs in UNG(+/+) cells. Furthermore, DSBs in UNG(+/+) and UNG(-/-) cells occur at distinct genomic loci, supporting differential mechanisms of DSB formation in UNG-competent and UNG-deficient cells. UNG(-/-) cells also showed increased evidence of replication fork instability (PCNA dispersal) when exposed to pemetrexed. Thymidine co-treatment rescues S-phase arrest in both UNG(+/+) and UNG(-/-) cells treated with IC50-level pemetrexed. However, following pemetrexed exposure, UNG(-/-) but not UNG(+/+) cells are refractory to thymidine rescue, suggesting that deficient uracil excision rather than dTTP depletion is the barrier to cell cycle progression in UNG(-/-) cells. Based on these findings we propose that pemetrexed-induced uracil misincorporation is genotoxic, contributing to replication fork instability, DSB formation and ultimately cell death.

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/µg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.

Removal of uracil by uracil DNA glycosylase limits pemetrexed cytotoxicity: overriding the limit with methoxyamine to inhibit base excision repair.

Uracil DNA glycosylase (UDG) specifically removes uracil bases from DNA, and its repair activity determines the sensitivity of the cell to anticancer agents that are capable of introducing uracil into DNA. In the present study, the participation of UDG in the response to pemetrexed-induced incorporation of uracil into DNA was studied using isogenic human tumor cell lines with or without UDG (UDG(+/+)/UDG(-/-)). UDG(-/-) cells were very sensitive to pemetrexed. Cell killing by pemetrexed was associated with genomic uracil accumulation, stalled DNA replication, and catastrophic DNA strand breaks. By contrast, UDG(+/+) cells were >10 times more resistant to pemetrexed due to the rapid removal of uracil from DNA by UDG and subsequent repair of the resultant AP sites (abasic sites) via the base excision repair (BER). The resistance to pemetrexed in UDG(+/+) cells could be reversed by the addition of methoxyamine (MX), which binds to AP sites and interrupts BER pathway. Furthermore, MX-bound AP sites induced cell death was related to their cytotoxic effect of dual inactivation of UDG and topoisomerase IIα, two genes that are highly expressed in lung cancer cells in comparison with normal cells. Thus, targeting BER-based therapy exhibits more selective cytotoxicity on cancer cells through a synthetic lethal mechanism.

Prospective study of one- vs two-unit umbilical cord blood transplantation following reduced intensity conditioning in adults with hematological malignancies.

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.

DNA repair modulates the vulnerability of the developing brain to alkylating agents.

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag(-/-)) or O(6)-methylguanine methyltransferase (Mgmt(-/-)), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt(-/-) neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag(-/-) neurons were for the most part significantly less sensitive than wild type or Mgmt(-/-) neurons to MAM and HN2. Aag(-/-) neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt(-/-) mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM-treated Aag(-/-) or MGMT-overexpressing (Mgmt(Tg+)) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in Mgmt(Tg+) mice treated with HN2. Collectively, these in vitro and in vivo studies demonstrate that the type of DNA lesion and the efficiency of DNA repair are two important factors that determine the vulnerability of the developing brain to long-term injury by a genotoxicant.

Time-dependent effect of non-Hodgkin's lymphoma grade on disease-free survival of relapsed/refractory patients treated with high-dose chemotherapy plus autotransplantation.

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.

Pre-mobilization therapy blood CD34+ cell count predicts the likelihood of successful hematopoietic stem cell mobilization.

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.

DNA repair in stem cell maintenance and conversion to cancer stem cells.

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.

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection.

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.

Hematopoietic stem cell gene therapy: progress toward therapeutic targets.

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.

Marked inactivation of O6-alkylguanine-DNA alkyltransferase activity with protracted temozolomide schedules.

Temozolomide, an oral DNA methylator that inactivates the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGAT), has demonstrated anticancer activity on protracted schedules. Protracted schedules may lead to an 'autoenhancement' of temozolomide's inherent cytotoxic potential by cumulative reduction of the cell's capacity for AGAT-mediated DNA repair and resistance. This study was undertaken to characterise AGAT inactivation and regeneration in the peripheral blood mononuclear cells (PBMCs) of patients treated on two protracted temozolomide schedules. O(6)-alkyl guanine-DNA alkyltransferase activity was measured in the PBMCs of patients treated on two phase I protracted temozolomide studies. Patients were treated daily for either 7 days every 2 weeks (Schedule A) or 21 days every 4 weeks (Schedule B). The effects of various temozolomide doses (75-175 mg m(-2)), treatment duration (7-21 days), and temozolomide plasma levels on AGAT inactivation and regeneration, as well as the relation between AGAT inactivation and toxicity, were studied. O(6)-alkyl guanine-DNA alkyltransferase activity in PBMCs was measured serially in 52 patients. Marked inactivation of AGAT occurred following 7 days of temozolomide treatment, with mean AGAT activity decreasing by 72% (P<0.0001). Similarly, mean AGAT activity decreased by 63 and 73% after 14 and 21 days of treatment, respectively (P<0.001 for both comparisons). O(6)-alkyl guanine-DNA alkyltransferase inactivation was greater after 7 days of treatment with higher doses of temozolomide than lower doses and remained markedly reduced 7 days post-treatment. However, AGAT inactivation following temozolomide treatment for 14 and 21 days was similar at all doses. On the continuous 21-day schedule, AGAT inactivation was significantly greater in patients who experienced severe thrombocytopenia than those who did not (90.3+/-5.5 vs 72.5+/-16.1%, P<0.045). In conclusion, protracted administration of temozolomide, even at relatively low daily doses, leads to significant and prolonged depletion of AGAT activity, which may enhance the antitumour activity of the agent.

Allogeneic mesenchymal stem cell infusion for treatment of metachromatic leukodystrophy (MLD) and Hurler syndrome (MPS-IH).

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.

Methoxyamine potentiates DNA single strand breaks and double strand breaks induced by temozolomide in colon cancer cells.

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.

MMTV promoter hypomethylation is linked to spontaneous and MNU associated c-neu expression and mammary carcinogenesis in MMTV c-neu transgenic mice.

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.

G156A MGMT-transduced human mesenchymal stem cells can be selectively enriched by O6-benzylguanine and BCNU.

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.

Sequential tumor biopsies in early phase clinical trials of anticancer agents for pharmacodynamic evaluation.

PURPOSE: 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. EXPERIMENTAL DESIGN: 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. RESULTS: 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). CONCLUSIONS: 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.

Overexpression of human O6-alkylguanine DNA alkyltransferase (AGT) prevents MNU induced lymphomas in heterozygous p53 deficient mice.

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.

Differential degradation rates of inactivated alkyltransferase in blood mononuclear cells and tumors of patients after treatment with O(6)-benzylguanine.

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.

Temozolomide: the effect of once- and twice-a-day dosing on tumor tissue levels of the DNA repair protein O(6)-alkylguanine-DNA-alkyltransferase.

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.