Primary analysis of a prospective, randomized, single-blinded phase II trial evaluating the HER2 peptide AE37 vaccine in breast cancer patients to prevent recurrence.

BACKGROUND: AE37 is the Ii-Key hybrid of the MHC class II peptide, AE36 (HER2 aa:776-790). Phase I studies showed AE37 administered with granulocyte macrophage colony-stimulating factor (GM-CSF) to be safe and highly immunogenic. A prospective, randomized, multicenter phase II adjuvant trial was conducted to evaluate the vaccine's efficacy. METHODS: Clinically disease-free node-positive and high-risk node-negative breast cancer patients with tumors expressing any degree of HER2 [immunohistochemistry (IHC) 1-3+] were enrolled. Patients were randomized to AE37 + GM-CSF versus GM-CSF alone. Toxicity was monitored. Clinical recurrences were documented and disease-free survival (DFS) analyzed. RESULTS: The trial enrolled 298 patients; 153 received AE37 + GM-CSF and 145 received GM-CSF alone. The groups were well matched for clinicopathologic characteristics. Toxicities have been minimal. At the time of the primary analysis, the recurrence rate in the vaccinated group was 12.4% versus 13.8% in the control group [relative risk reduction 12%, HR 0.885, 95% confidence interval (CI) 0.472-1.659, P = 0.70]. The Kaplan-Meier estimated 5-year DFS rate was 80.8% in vaccinated versus 79.5% in control patients. In planned subset analyses of patients with IHC 1+/2+ HER2-expressing tumors, 5-year DFS was 77.2% in vaccinated patients (n = 76) versus 65.7% in control patients (n = 78) (P = 0.21). In patients with triple-negative breast cancer (HER2 IHC 1+/2+ and hormone receptor negative) DFS was 77.7% in vaccinated patients (n = 25) versus 49.0% in control patients (n = 25) (P = 0.12). CONCLUSION: The overall intention-to-treat analysis demonstrates no benefit to vaccination. However, the results confirm that the vaccine is safe and suggest that vaccination may have clinical benefit in patients with low HER2-expressing tumors, specifically TNBC. Further evaluation in a randomized trial enrolling TNBC patients is warranted.

Recognition of Ii-Key/MHC class II epitope hybrids derived from proinsulin and GAD peptides by T cells in type 1 diabetes.

In order to determine whether the Ii-Key technology can enhance the presentation of specific epitopes associated with type 1 diabetes, we have designed and synthesized a series of Ii-Key/proinsulin and GAD epitope hybrid peptides. Peptides of proinsulin and GAD shown to be recognized by CD4+ T cells of type 1 diabetes patients have been selected from the literature and modified with Ii-Key. A total of 23 Caucasian type 1 diabetes subjects and 17 normal subjects as controls were included in the study. Reactive T cells were identified using an IFN-γ ELISPOT assay. We selected 5 proinsulin and 5 GAD epitopes. Regarding the activity of the proinsulin Ii-Key hybrids, 3 out of 15 patients (20%) demonstrated a positive response to one or more Ii-Key hybrid peptides compared to no responders in the control subjects. Two out of 8 patients demonstrated a positive response to one or more Ii-Key/GAD65 hybrids. Proinsulin Ii-Key hybrids and peptides were recognized only by DR3/DR4 0302+ve diabetic patients. Control subjects showed no detectable response to stimulation with Ii-Key hybrids or peptides, neither for proinsulin nor GAD65. We have now shown that the use of Ii-Key-modified MHC class II epitopes, derived from proteins associated with insulin-secreting cells, can detect the presence of specifically activated CD4+ T helper cells with greater sensitivity than unmodified epitopes in the standard ELISPOT assay. The use of these technologies may be of use in identifying patients at the earliest stages of type 1 diabetes.

Distinct characteristics of seasonal (Bet v 1) vs. perennial (Der p 1/Der p 2) allergen-specific CD4(+) T cell responses.

BACKGROUND: A better understanding of allergen-specific CD4(+) T cell responses is needed to help improving immunological therapies. Objective To compare CD4(+) T cell responses against seasonal (Bet v 1) and perennial (Der p 1, Der p 2) allergens. METHODS: Major histocompatibility complex class II peptide tetramers were engineered to monitor allergen-specific T cell responses. After in vitro expansion, tetramer(+) cells were tested for surface markers using cytofluorometry. Cytokine gene expression and production were assessed using quantitative PCR and cytokine surface capture assays, respectively. RESULTS: Tetramer(+) cells were detected in 19 patients allergic to house dust mites (HDM), seven allergic to birch pollen, 13 allergic to both and nine non-allergics with either an HLA-DRB1(*) 0101, (*) 0301, (*) 1501 or an HLA-DPB1(*) 0401 background. High-avidity T cells are elicited against the immunodominant Bet v 1(141-155) epitope, whereas broader low-avidity T cell responses are induced against Der p 1(16-30) ,(110-124) ,(171-185) and Der p 2(26-40,107-121) epitopes. Responses against Bet v 1 involve effector (CDL62 low, CCR7 low) or central (CD62L(+) , CCR7(+) ) memory cells in allergic and non-allergic individuals, respectively, whereas central memory cells are mostly detected against mite allergens. In non-allergics, both mite and Bet v 1-specific T cells produce IFN-γ and IL-10. In contrast to Bet v 1-driven Th2 responses, mite allergens induce highly polymorphic responses in allergics, including Th1, Th2/Th17 or mixed Th1/Th2 profiles. Mite-specific T cell frequencies in the blood remain in the range of 1-6 × 10(-4) CD4(+) T cells throughout the year. CONCLUSION: Different memory CD4(+) T cell responses are elicited in the context of chronic vs. seasonal stimulation with the allergen(s). The heterogeneity in the patterns of CD4(+) T cell responses observed in patients allergic to HDMs should be taken into account for specific immunotherapy.

MHC class II allosteric site drugs: new immunotherapeutics for malignant, infectious and autoimmune diseases.

The discovery of the interactions of the 'Ii-Key' segment of the Ii protein with the major histocmpatibility complex (MHC) Class II allosteric site, which is adjacent to the antigenic peptide-binding site, creates therapeutic opportunities by regulating the antigenic peptide binding to MHC class II molecules. The binding of Ii-Key to the MHC class II allosteric site loosens the hold of the MHC Class II 'clamshell' on antigenic peptides and leads to highly efficient antigenic peptide charging to or releasing from the MHC class II antigenic peptide-binding groove. Ii-Key peptide-induced spilling of bound antigenic peptide, or replacement with inert blockers, leads to 'inert immunosuppression'. Highly efficient replacement of ambient with vaccine peptides by Ii-Key permits 'active immunosuppression' for antigen-specific control of autoimmune diseases in the absence of cytokines or adjuvants. On the other hand, active immunization against cancer or infectious disease can result from epitope replacement mediated by Ii-Key and accompanied by cytokines or other adjuvants. Finally, linking the Ii-Key peptide through a simple polymethylene bridge to an antigenic sequence vastly increases the potency of MHC Class II peptide vaccines. In summary, the discovery of the MHC class II allosteric site allows one to increase the efficiency of MHC class II-related, antigenic epitope-specific therapy for malignant, infectious, and autoimmune diseases. The focus of this review is on the mechanism and potential clinical use of such novel allosteric site-directed, Ii-key drugs.

Increasing the potency of MHC class II-presented epitopes by linkage to Ii-Key peptide.

We previously found that peptide Ii77-92 from the immunoregulatory Ii protein significantly enhances the binding of antigenic peptides to MHC class II molecules. Now a series of hybrids have been constructed linking LRMK, the active core region of the Ii77-92 peptide, to an antigenic epitope of cytochrome C. In vitro T cell hybridoma stimulation by some of these hybrids is up to 250 times more potent than by the antigenic peptide. The biological activities of the hybrids were tested in terms of length and composition of the linker. Simple spacers containing a polymethylene bridge (-HN-CH(2)-CH(2)-CH(2)-CH(2)-CO(2)-) were fully active in these hybrids which can enhance vaccination with MHC class II-presented epitopes.

Genetic modulation of tumor antigen presentation.

An effective cancer-cell vaccine is created by expressing major-histocompatibility-complex (MHC) class II molecules without the invariant chain protein (Ii) that normally blocks the antigenic-peptide-binding site of MHC class II molecules at their synthesis in the endoplasmic reticulum. Such tumor-cell constructs are created either by the transfer of genes for MHC class IIalpha and beta chains, or by the induction of MHC class II molecules and Ii protein with a transacting factor, followed by Ii suppression using antisense methods. Preclinical validation of this approach is reviewed with the goal of using this immunotherapy for metastatic human cancers.

Generation of a nude mouse tumor model for in vivo replication of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a common opportunistic infection resulting in retinitis in 15%-40% of AIDS patients. Several anti-HCMV therapies are currently available, and new treatments are in various stages of development. An HCMV animal model involving in vivo infection of human cells without the dependence on human fetuses or multiple surgical procedures has been developed. A human glioblastoma cell line that is permissive for HCMV replication (U373MG) was adapted to grow as a subcutaneous tumor in nude mice. These tumors arise in approximately 7 days and grow progressively. An evaluation of HCMV DNA replication demonstrated an increase in the accumulation of HCMV DNA within infected tumors from 48 to 168 h after infection. Immunohistochemical analysis showed focal areas of HCMV infection in which expression of immediate-early and late antigens was detected. In addition, it was demonstrated that ganciclovir inhibited HCMV DNA replication in vivo in a dose-dependent manner.

Pharmacokinetics and tissue distribution of a DNA-methyltransferase antisense (MT-AS) oligonucleotide and its catabolites in tumor-bearing nude mice.

The pharmacokinetics of a 20-mer phosphorothioate antisense oligodeoxynucleotide was investigated in nude mice bearing a s.c. human lung carcinoma. The oligodeoxynucleotide, referred to as DNA-methyltransferase antisense (MT-AS) was designed to bind to the mRNA that coded for DNA-methyltransferase, an enzyme that controls the extent of methylation of 5'-cytosine. MT-AS was administered at four different doses (10, 30, 100 and 300 mg/kg) as an i.v. bolus in a composite study design. A maximum of four blood samples were collected from any single animal, followed by sacrifice to obtain tissues. The plasma and tissue samples were collected from 5 min to 48 h after dosing and were processed by anion-exchange HPLC (high performance liquid chromatography) and by capillary gel electrophoresis. On the basis of total (i.e., 15-mer to 20-mer species) MT-AS plasma concentrations as determined by HPLC, total clearance ranged from 7.9 ml/min/kg at the 30-mg/kg dose level to 15.2 ml/min/kg at 10 mg/kg; however, there were no definitive dose-dependent changes in clearance. The volume of distribution at steady state increased from a low value of 379 ml/kg at 30 mg/kg to a high of 1983.0 ml/kg at 300 mg/kg, a result that suggests saturable protein binding. In vitro plasma protein binding data supported this possibility, because the percentage of MT-AS bound decreased at high MT-AS concentrations. MT-AS distributed into most tissues, with a general rank order of kidney > liver > tumor > lung > muscle > brain. Analysis of plasma samples by capillary gel electrophoresis from 2 h to 8 h revealed that about 50% of the total oligodeoxynucleotides were due to the parent 20-mer MT-AS; the remainder consisted of 15-mer to 19-mer catabolites. Of particular interest was the relatively high tumor uptake of MT-AS. These results will support future studies designed to characterize the pharmacological action of MT-AS and its efficacy in preclinical models.

Sensitization of multidrug-resistant human leukemia cells with MDR1-targeted antisense and inhibition of drug-mediated MDR1 induction.

Increased expression of MDR1 is strongly implicated in the appearance of chemotherapeutic drug resistance in cancer, especially hematological malignancies. We therefore examined the potential of antisense oligonucleotides to inhibit MDR1 and restore sensitivity to drug-resistant human lymphoblastic cells (CCRF-CEM). Treatment with two different phosphorothioate-modified antisense sequences as well as a DNA-RNA hybrid sequence resulted in a 30 to 45% decrease in MDR1 expression as determined by staining with the monoclonal antibody MRK16 followed by flowcytometry (FCM) analysis. Further, inhibition of MDR1 expression persisted for 3 days after removal of oligonucleotides. Increased accumulation of rhodamine 123 and nearly a three-fold sensitization of cells to vincristine paralleled the reduction in staining with MRK16. Reversed or scrambled control sequences had no effect in any of the assays. During the course of these studies, we observed a 25 to 75% increase in MRK16 staining of cells treated with the chemotherapeutic agents daunorubicin and vincristine as well as by the resistance reversal agents verapamil and cyclosporin. Treatment of cells with antisense oligonucleotides prior to exposure to daunorubicin or cyclosporin reduced the increase in MRK16 staining. These results indicate that antisense targeted to MDR1 can sensitize drug-resistant leukemia cells and suggest that antisense treatment may prevent the emergence of MDR1-mediated drug resistance.

The relationship of a novel drug-resistant phenotype in C3H10T1/2 cells selected with alkylating agents to neoplastic transformation and ATP metabolism.

We investigated the resistance to alkylating agents in parental, drug-selected and neoplastically transformed C3H10T1/2 (10T1/2) murine fibroblasts. Similar levels of resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were observed in cells selected for resistance to MNNG or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) as well as in cells transformed by a single treatment with MNNG. Surprisingly, neither the levels of O6-alkylguanine-DNA alkyltransferase (AT) nor glutathione-S-transferase (GST) were altered in drug-resistant cells. In contrast, changes in ATP metabolism were observed in both transformed and MNNG-selected cells after treatment with MNNG. Specifically, 3 h after treatment with 5 microg/ml MNNG, ATP levels decreased by 85% and 74% in MNNG-selected and transformed cells, respectively, compared to only a 28% decrease in parental cells. Therefore, rather than contributing to cell sensitivity to alkylating agents, the ability to rapidly utilize ATP and tolerate resulting decreases in ATP levels may in some cases play a role in protection from the cytotoxic effects of alkylating agents.

Inhibition of tumorigenesis by a cytosine-DNA, methyltransferase, antisense oligodeoxynucleotide.

This paper tests the hypothesis that cytosine DNA methyltransferase (DNA MeTase) is a candidate target for anticancer therapy. Several observations have suggested recently that hyperactivation of DNA MeTase plays a critical role in initiation and progression of cancer and that its up-regulation is a component of the Ras oncogenic signaling pathway. We show that a phosphorothioate-modified, antisense oligodeoxynucleotide directed against the DNA MeTase mRNA reduces the level of DNA MeTase mRNA, inhibits DNA MeTase activity, and inhibits anchorage independent growth of Y1 adrenocortical carcinoma cells ex vivo in a dose-dependent manner. Injection of DNA MeTase antisense oligodeoxynucleotides i.p. inhibits the growth of Y1 tumors in syngeneic LAF1 mice, reduces the level of DNA MeTase, and induces demethylation of the adrenocortical-specific gene C21 and its expression in tumors in vivo. These results support the hypothesis that an increase in DNA MeTase activity is critical for tumorigenesis and is reversible by pharmacological inhibition of DNA MeTase.

Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

Relationship between O6-alkylguanine-DNA alkyltransferase activity and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation, transformation, and cytotoxicity in C3H/10T1/2 cells expressing exogenous alkyltransferase genes.

While a great deal of evidence has directly implicated the importance of O6-alkylation of guanine in the mutagenicity of alkylating agents, evidence demonstrating the oncogenic potential of this lesion has been largely indirect. We have combined a well-studied in vitro neoplastic transformation system (using C3H/10T1/2 mouse cells) with a proven method of gene transfection for expressing the bacterial O6-alkylguanine-DNA alkyltransferase (AT; EC 2.1.1.63) repair genes ada and ogt to generate subclones which possess augmented repair capability toward specific DNA lesions. The products of these genes specifically and differentially repair O6-methylguanine (O6-MeGua), O4-methylthymine (O4-MeThy), and methylphosphotriesters. We show that the level of expression of either the ada or the ogt AT gene in C3H/10T1/2 cells directly correlates with protection against mutation to ouabain resistance by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Subclones expressing 70 fmol of AT per 10(6) cells exhibited a mutation frequency approximately 1/40th of that of clones expressing 15 fmol of AT per 10(6) cells when treated with MNNG at 0.4 micrograms/ml. Protection against mutagenesis by MNNG at 0.8 micrograms/ml, however, did not exceed 12-fold even in subclones expressing greater than 100 fmol of AT per 10(6) cells. As an MNNG dose of 0.6 micrograms/ml was sufficient to saturate more than 95% of the AT activity in any of the clones, the residual mutation frequency may have been caused by unrepaired O6MeGua lesions. In contrast to mutagenesis, protection against neoplastic transformation in vitro, in cells expressing high levels of AT, was most pronounced in cells treated with the highest dose of MNNG used (1.2 micrograms/ml). Low levels of transformation caused by MNNG at 0.4 and 0.8 micrograms/ml were not consistently inhibited in those clones. These data suggest that O6-MeGua formation is of major but not unique significance in the neoplastic transformation of C3H/10T1/2 cells by MNNG.

Inhibition of N-nitrosomethylbenzylamine-induced esophageal neoplasms by the Bowman-Birk protease inhibitor.

Model systems in which carcinogenesis by given agents can be prevented or reduced offer a means of gaining insight into the mechanism(s) of action of carcinogens and the feasibility of chemoprevention in humans. In the current study, the ability of the soy-bean derived Bowman-Birk protease (BBI) to suppress esophageal carcinogenesis induced by N-nitrosomethylbenylamine (NMBzA) was examined. Esophageal lesions were produced in male Sprague-Dawley rats by i.p. injection of 2 mg/kg NMBzA twice weekly for 3 weeks. Groups receiving BBI were fed three tablets a week containing 180 mg BBI each in a mixture of Witepsol H15 and peanut butter for the duration of the experiment. The frequency of papillomas and carcinomas was reduced 45% in groups receiving BBI. Furthermore, the frequency of appearance of five separate characteristics of preneoplastic lesions was significantly reduced in the esophagi of BBI-treated animals. The most significant reduction was in the total number of lesions with simple hyperplasia. Groups receiving NMBzA and placebo tablets, containing only Witepsol H15 and peanut butter, did not display statistically significant differences in the frequency of esophageal lesions as compared to animals receiving NMBzA alone. These results demonstrate that BBI can effectively inhibit NMBzA-induced esophageal tumors when given in tablet form separate from the regular diet.

X-ray induction of O6-alkylguanine-DNA alkyltransferase protects against some of the biological effects of N-methyl-N'-nitro-N-nitrosoguanidine in C3H 10T1/2 cells.

We have shown previously that the repair of O6-methylguanine can be induced in murine fibroblasts (C3H 10T1/2 cells) by exposure to X rays. The magnitude of the response is less, however, than is observed in the well-characterized adaptive response of various prokaryotes to methylating agents. To determine whether the induction of O6-alkylguanine-DNA alkyltransferase in C3H 10T1/2 cells is sufficient for protection against the genotoxic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), cells were challenged with MNNG after alkyltransferase induction by 1.5 Gy X rays and assayed for cytotoxicity, mutagenicity, and neoplastic transformation. Preirradiated cells were significantly more resistant to the mutagenic effects of MNNG as scored by formation of ouabain-resistant colonies. The protective effect was greatest in cells challenged with a low dose (0.2 or 0.4 micrograms/ml) of MNNG. Protection against neoplastic transformation by MNNG was also observed, although the protective effect in this case was significant only in cells treated with a high dose (1.0 micrograms/ml) of MNNG. In cells that were preirradiated, there was no reduction in the cytotoxicity caused by MNNG or the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These data indicate that alkyltransferase induction in C3H 10T1/2 cells is sufficient to protect cells against some of the genotoxic effects of the alkylating agent MNNG. The data also suggest that formation of O6-alkylguanine may not be the only means by which alkylating agents can transform C3H 10T1/2 cells.

Beta-deuteration of N-nitrosoethylmethylamine causes a shift in DNA methylation from rat liver to esophagus.

While N-nitrosoethylmethylamine (NEMA) is carcinogenic primarily for the liver, its beta-trideuterated derivative, N-nitroso( [2-D3]ethyl)methylamine (NEMA-d3), also produces a high incidence of tumors in the esophagus. To determine whether this shift in organ specificity is associated with an altered pattern of DNA alkylation, [methyl-14C]- and [1-ethyl-14C]-labeled NEMA-d3 were administered to adult male Fischer 344 rats as a single i.p. dose (0.05 mmol/kg; 4 h survival). Levels of methylated and ethylated purines in the DNA of various organs were determined by radio-chromatography on Sephasorb-HP columns. When compared to previous data using undeuterated NEMA, 7-methylguanine levels were found to be reduced by approximately 30% in liver and kidney, but were 160% greater in esophagus. This resulted in a decrease in the 7-methylguanine ratio for liver/esophagus from 109 to 29. O6-Methylguanine was diminished in liver and kidney, but levels in lung and esophagus were too low for quantitative detection. Similarly, deuteration led to an 18% decrease of 7-ethylguanine in hepatic DNA. The observed increase in esophageal DNA methylation correlates with the increased carcinogenicity of NEMA-d3 relative to undeuterated NEMA in that organ. Since pharmacokinetic studies have shown that beta-trideuteration of NEMA does not alter its bioavailability, the data suggest that the observed shift in target organ results from isotopically-induced changes in the balance among competing metabolic pathways in different rat tissues.

Modulation of mouse fibroblast adipocyte differentiation by X-rays and TPA.

The purpose of this study was to determine whether X-rays and the tumor promoter TPA which, in combination, are more effective at transforming cells in vitro than is either alone, may also be more effective at inhibiting differentiation in combination. Previous studies with 3T3 T proadipocytes have shown that both the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA) and a classical initiator, UV light, can inhibit differentiation. We have now extended these studies by examining the effects of X-rays and TPA administered singly and in combination on differentiation of 3T3 T proadipocytes caused by incubation in medium containing 25% human plasma and on 5-azacytidine induced differentiation of C3H/10T1/2 cells. X-rays alone caused a dose-dependent inhibition of differentiation of 3T3 proadipocytes from 1.0 to 9.0 Gy. Low doses of X-rays (1.0 Gy) also inhibited differentiation of C3H/10T1/2 cells treated with 5-azacytidine but high doses (6.0 Gy) actually caused differentiation in the absence of 5-azacytidine treatment. TPA also inhibited differentiation of both 3T3 T and 5-azacytidine-treated C3H/10T1/2 cells at a dose of 0.1 micrograms/ml. At the lower dose of 0.001 micrograms/ml TPA, 3T3 T differentiation was inhibited only 10%, while 2.5 Gy of X-rays inhibited differentiation approximately 20%. Combined treatment of cells with X-rays and TPA caused more than 60% inhibition of differentiation, i.e., approximately twice the inhibition that would be expected if the effects of these two agents were additive. This synergistic effect suggests that in some systems there may be a closer association between the multistage process of neoplastic transformation and the inhibition of processes involved in cell differentiation than previous experiments, performed with initiators or promoters administered separately, have indicated.

Inhibition of X-ray-induced exencephaly by protease inhibitors.

We report that protease inhibitors can reduce the incidence of radiation-induced exencephaly in mice. Previous studies from this and other laboratories have demonstrated that protease inhibitors, in particular antipain and the Bowman-Birk inhibitor, are effective anticarcinogenic agents in a variety of in vivo systems and inhibit cell transformation in vitro. Given our results, further investigation is warranted into preventive effects of protease inhibitors on the inhibition of neural tube defects.

Single-dose toxicokinetics of N-nitrosomethylethylamine and N-nitrosomethyl (2,2,2-trideuterioethyl)amine in the rat.

To investigate the origins of an organotropic shift toward increasing esophageal carcinogenicity and DNA alkylation caused by beta-trideuteration of the hepatocarcinogen, N-nitrosomethylethylamine (NMEA), the single-dose toxicokinetics of NMEA and N-nitrosomethyl(2,2,2-trideuterioethyl)amine (NMEA-d3) has been characterized in 8-week-old male Fischer 344 rats by analysis using high performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to rats revealed biphasic first order elimination with a terminal half-life of 9.46 +/- 0.69 min for unchanged NMEA and 28.9 +/- 2.4 min for total radioactivity. Extensive conversion to polar metabolites was observed in the chromatograms. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMEA were 39.9 +/- 4.6 ml/min/kg and 496 +/- 36 ml/kg, respectively. There was negligible plasma protein binding and no detectable NMEA was excreted unchanged in the urine. Larger doses given by gavage indicated a systemic bioavailability of 25 +/- 1%. Similar doses of NMEA-d3 given to other groups of rats revealed no significant differences in any of the toxicokinetic parameters. No N-nitrosomethyl(2-hydroxyethyl)amine was found as a detectable metabolite of NMEA or NMEA-d3 in any of the blood or urine samples which were analyzed. When considered together, the data suggest that previously observed differences in organ specificity for the carcinogens, NMEA and NMEA-d3, are not due to differences in the total amounts of nitrosamine reaching particular tissues, but may have other localized causes such as differences in the enzymes responsible for metabolism which are present in each tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

O6-alkylguanine-DNA alkyltransferase and sensitivity to procarbazine in human brain-tumor xenografts.

The level of O6-alkylguanine-deoxyribonucleic acid (DNA) alkyltransferase (AT) was determined in 15 human brain-tumor xenografts in athymic mice. This enzyme is a primary intracellular repair mechanism for lesions produced at the O6 position of guanine by a wide range of alkylating agents, including nitrosoureas and procarbazine. Its activity ranged from undetectable in five tumor lines to 2338 fmol/mg protein in N-1941, a human glioblastoma xenograft. The sensitivity of 10 of these xenografts to procarbazine was determined and it was found that four of the five tumor lines with AT levels of more than 100 fmol/mg protein had growth delays after procarbazine treatment of less than 20 days, whereas all five lines with undetectable AT levels had growth delays of over 30 days. The primary cytotoxic DNA adduct produced by procarbazine (namely, O6-methylguanine) was found to be significantly higher in two sensitive lines with low AT levels than in a highly resistant line with a high AT level. These data suggest that the AT levels of individual brain tumors can be used as predictive indicators of their susceptibility to drugs that exert their antineoplastic effect primarily by O6-alkylation of guanine in nuclear DNA.