Genetic variants of kinase suppressors of Ras (KSR1) to predict survival in patients with ERα-positive advanced breast cancer.

In patients with breast cancer (BC), deregulation of estrogen receptor (ERα) activity may account for most resistance to endocrine therapies. Our previous study used a whole-human kinome siRNA screen to identify functional actors in ERα modulation and showed the implication of proteins kinase suppressors of ras (KSR1). From those findings we evaluated the clinical impact of KSR1 variants in patients with ERα+ BC treated with TAM. DNA was obtained from 222 patients with advanced ERα+ BC treated with TAM who had undergone surgery from 1981 to 2003. We selected three potentially functional relevant KSR1 polymorphisms; two within the 3'UTR (rs224190, rs1075952) and one in the coding exon 7 (rs2293180). The primary end points were overall survival (OS) and disease-free survival (DFS). After a 6.4-year median follow-up, patients carrying the rs2241906 TT genotype showed shorter DFS (2.1 vs 7.1 years, P=0.005) and OS (2.6 vs 8.4 years P=0.002) than those with the TC or TT genotypes. Those associations remained significant in the multivariable analysis adjusting age, lymph node status, LMTK3 and IGFR variants and HER2 status. The polymorphisms rs2241906 and rs1075952 were in linkage disequilibrium. No association was shown between rs2293180 and survival. Among the actors of ERα signaling, KSR1 rs2241906 variants may predict survival in patients with advanced ERα+ BC treated with adjuvant TAM.

Intratumoral expression profiling of genes involved in angiogenesis in colorectal cancer patients treated with chemotherapy plus the VEGFR inhibitor PTK787/ZK 222584 (vatalanib).

The phase III CONFIRM clinical trials demonstrated that metastatic colorectal cancer patients with elevated serum lactate dehydrogenase (LDH) had improved outcome when the vascular endothelial growth factor receptor (VEGFR) inhibitor PTK/ZK (Vatalanib) was added to FOLFOX4 chemotherapy. We investigated the hypothesis that high intratumoral expression of genes regulated by hypoxia-inducible factor-1 alpha (HIF1α), namely LDHA, glucose transporter-1 (GLUT-1), VEGFA, VEGFR1, and VEGFR2, were predictive of outcome in CONFIRM-1. Tumor tissue was isolated by laser-capture microdissection from 85 CONFIRM-1 tumor specimens; FOLFOX4/placebo n=42, FOLFOX4/PTK/ZK n=43. Gene expression was analyzed using quantitative RT-PCR. In univariate analyses, elevated mRNA expression of LDHA, GLUT-1, and VEGFR1 were associated with response to FOLFOX4/PTK/ZK. In univariate and multivariate analyses, elevated LDHA and VEGFR1 mRNA levels were associated with improved progression-free survival in FOLFOX4/PTK/ZK patients. Furthermore, increased HIF1α and VEGFR2 mRNA levels were associated with decreased survival in FOLFOX/placebo patients but not in patients who received FOLFOX4/PTK/ZK. These are the first data suggesting intratumoral mRNA expression of genes involved in angiogenesis/HIF pathway may predict outcome to VEGFR-inhibitors. Biomarkers that assist in directing VEGFR-inhibitors toward patients with an increased likelihood of benefit will improve the cost-effectiveness of these promising agents.

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis. METHODS: A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth. RESULTS: Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis. CONCLUSION: Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

The Cyclin D1 (CCND1) A870G polymorphism predicts clinical outcome to lapatinib and capecitabine in HER2-positive metastatic breast cancer.

BACKGROUND: Lapatinib plus capecitabine emerged as an efficacious therapy in metastatic breast cancer (mBC). We aimed to identify germline single-nucleotide polymorphisms (SNPs) in genes involved in capecitabine catabolism and human epidermal receptor signaling that were associated with clinical outcome to assist in selecting patients likely to benefit from this combination. PATIENTS AND METHODS: DNA was extracted from 240 of 399 patients enrolled in EGF100151 clinical trial (NCT00078572; clinicaltrials.gov) and SNPs were successfully evaluated in 234 patients. The associations between SNPs and clinical outcome were analyzed using Fisher's exact test, Kaplan-Meier curves, log-rank tests, likelihood ratio test within logistic or Cox regression model, as appropriate. RESULTS: There were significant interactions between CCND1 A870G and clinical outcome. Patients carrying the A-allele were more likely to benefit from lapatinib plus capecitabine versus capecitabine when compared with patients harboring G/G (P = 0.022, 0.024 and 0.04, respectively). In patients with the A-allele, the response rate (RR) was significantly higher with lapatinib plus capecitabine (35%) compared with capecitabine (11%; P = 0.001) but not between treatments in patients with G/G (RR = 24% and 32%, respectively; P = 0.85). Time to tumor progression (TTP) was longer in patients with the A-allele treated with lapatinib plus capecitabine compared with capecitabine (median TTP = 7.9 and 3.4 months; P < 0.001), but not in patients with G/G (median TTP = 6.1 and 6.6 months; P = 0.92). CONCLUSION: Our findings suggest that CCND1A870G may be useful in predicting clinical outcome in HER2-positive mBC patients treated with lapatinib plus capecitabine.

A distinct strategy to generate high-affinity peptide binders to receptor tyrosine kinases.

We describe a novel and general way of generating high affinity peptide (HAP) binders to receptor tyrosine kinases (RTKs), using a multi-step process comprising phage-display selection, identification of peptide pairs suitable for hetero-dimerization (non-competitive and synergistic) and chemical synthesis of heterodimers. Using this strategy, we generated HAPs with K(D)s below 1 nM for VEGF receptor-2 (VEGFR-2) and c-Met. VEGFR-2 HAPs bound significantly better (6- to 500-fold) than either of the individual peptides that were used for heterodimer synthesis. Most significantly, HAPs were much better (150- to 800-fold) competitors than monomers of the natural ligand (VEGF) in various competitive binding and functional assays. In addition, we also found the binding of HAPs to be less sensitive to serum than their component peptides. We believe that this method may be applied to any protein for generating high affinity peptide (HAP) binders.

Novel frameworks as a source of high-affinity ligands.

The past year has seen further maturation of the techniques used to display populations of proteins and peptides and to select members with desired properties. Many protein domains have now been displayed on genetic packages, diverse populations have been made, and binders with specific useful properties have been selected. Affinity maturation has been demonstrated so that binding in the low nanomolar to subnanomolar range by non-antibodies is now achievable.

Isolation, characterization, and recovery of small peptide phage display epitopes selected against viable malignant glioma cells.

Phage display techniques rely on nearly random oligonucleotide sequences inserted into the protein III filament binding protein of an Escherichia coli filamentous phage M13 to generate a library of phage that express more than 10(7) different peptides. Phage that expresses a sequence having high affinity for a specific molecule, cell, or tissue can then be isolated through selective binding and recovery. Selected phage cannot only be used as gene transfer vectors in themselves, but the small peptide epitopes can be sequenced and potentially recombined into the attachment proteins of viral vectors, or used by themselves to target other therapeutic agents and diagnostic imaging radiolabels. Most phage display selections are carried out against purified and/or fixed protein targets, raising concerns as to the relevance of the selected epitopes. We have selected phage from the CMTI library against viable U87-MG human malignant glioma cells using a derivation of biopanning. The library, which initially contained phage expressing 2x10(7) different epitope sequences, collapsed after four rounds of selection such that 42% of recovered clones expressed a consensus sequence. Selective binding to viable adherent U87-MG cells was subsequently demonstrated under physiologic conditions at 167% (+/-27%) unselected phage using a novel, viable enzyme-linked immunosorbent assay technique. In comparison, there was no difference in binding to control 9L rat gliosarcoma, PANC-1 human pancreatic adenocarcinoma, T98-MG human malignant glioma, or AST-4 human malignant glioma cells of selected compared to unselected phage. Using polymerase chain reaction, the epitope was recovered with flanking unique restriction sites for recombination into a herpes simplex virus type-1 vector. This study demonstrates and discusses optimized methodologies for using phage display to target viable cells.

The role of dUTPase and uracil-DNA repair in cancer chemotherapy.

Thymidylate metabolism is an important target for chemotherapeutic agents that combat a variety of neoplastic diseases including head and neck, breast and gastrointestinal cancers. Therapeutic strategies applied to this pathway target the thymidylate synthase (TS) reaction that catalyzes the reductive methylation of deoxyuridylate (dUMP) to form thymidylate (TMP). This reaction represents the sole de novo source of TMP required for DNA replication and repair. Inhibitors of this pathway include the widely utilized fluoropyrimide and antifolate classes of anti-cancer agents. Studies attempting to elucidate the molecular mechanisms of cell killing mediated by inhibitors of the TS reaction suggest that cytotoxicity results from a process known as "thymineless death". This term describes the extreme TTP pool depletion observed following TS inhibition. Although depletion of TTP pools is clearly involved in this process, there is now considerable evidence implicating aberrant uracil-DNA metabolism as an important mechanism of toxicity. Upon TS inhibition, dUTP pools may accumulate, inducing repeated cycles of uracil misincorporation into DNA and repair-mediated DNA damage. Central to the uracil-misincorporation pathway are the enzymes deoxyuridine nucleotidohydrolase (dUTPase) (EC 3.6.1.23) and uracil-DNA glycoslyase (UDG) (EC 3.2.2.3). dUTPase catalyzes the hydrolysis of dUTP to form dUMP and pyrophosphate thereby eliminating dUTP and preventing its utilization by DNA polymerases during replication and repair. UDG initiates the base excision repair pathway effectively removing any uracil residues that may arise in DNA. Under normal conditions, uracil is precluded from DNA by the combined actions of dUTPase and UDG. However, during TS inhibition, dUTP pools may accumulate and overwhelm dUTPase, resulting in repeated cycles of uracil misincorporation and detrimental repair leading to strand breaks and cell death. Because dUTPase plays a pivotal role in regulating cellular dUTP pools, this enzyme could have profound effects on the efficacy of agents that target thymidylate biosynthesis. This article reviews our current understanding of the role of aberrant uracil-DNA metabolism as a contributing mechanism of cytotoxicity initiated by chemotherapeutic agents that target de novo thymidylate metabolism. The role of dUTPase expression in modulating therapeutic response is presented including evidence from yeast and mammalian cell culture models and clinical studies. The regulation of human dUTPase isoforms in normal and neoplastic tissues will be reviewed as well as the role of dUTPase expression as a prognostic marker for overall survival and response to therapy in colon cancer.

Deoxyuridine triphosphatase (dUTPase) expression and sensitivity to the thymidylate synthase (TS) inhibitor ZD9331.

Uracil DNA misincorporation and misrepair of DNA have been recognized as important events accompanying thymidylate synthase (TS) inhibition. dUTPase catalyses the hydrolysis of dUTP to dUMP, thereby maintaining low intracellular dUTP. We have addressed the relationship between dUTPase expression and cellular sensitivity to TS inhibition in four human lung tumour cell lines. Sensitivity (5-day MTT assay) to the growth inhibitory effects of the non-polyglutamatable, specific quinazoline TS inhibitor ZD9331, varied up to 20-fold (IC(50)3-70 nM). TS protein expression correlated with TS activity (r(2)= 0.88, P = 0.05). Intracellular concentrations of drug following exposure to ZD9331 (1 microM, 24 h) varied by approximately 2-fold and dTTP pools decreased by > 80% in all cell lines. No clear associations across the cell lines between intracellular drug concentrations, TS activity/expression, or TTP depletion could be made. dUTPase activity varied 17-fold and correlated with dUTPase protein expression (r(2)= 0.94, P = 0.03). There was a striking variation in the amount of dUTP formed following exposure to ZD9331 (between 1.3 and 57 pmole 10(-6)cells) and was in general inversely associated with dUTPase activity. A large expansion in the dUTP pool was associated with increased sensitivity to a 24-h exposure to ZD9331 in A549 cells that have low dUTPase activity/expression. dUTPase expression and activity were elevated (approximately 3-fold) in two variants of a human lymphoblastoid cell line with acquired resistance to TS inhibitors, further suggesting an important role for this enzyme in TS inhibited cells.

dUTP nucleotidohydrolase isoform expression in normal and neoplastic tissues: association with survival and response to 5-fluorouracil in colorectal cancer.

Aberrant dUTP metabolism plays a significant role in the underlying molecular mechanisms of cell killing mediated by inhibitors of thymidylate biosynthesis. dUTP nucleotidohydrolase (dUTPase) is the key regulator of dUTP pools, and significant evidence exists suggesting that the expression of this enzyme may be an important determinant of cytotoxicity mediated by inhibitors of thymidylate synthase (TS). In this study, we have determined the expression patterns of dUTPase in normal and neoplastic tissues and examined the association between dUTPase expression and response to 5-fluorouracil (5-FU)-based chemotherapy and overall survival in colorectal cancer. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using a monoclonal antibody (MAb), DUT415, that cross-reacts with both nuclear and mitochondrial isoforms of human dUTPase. Nuclear and cytoplasmic staining was observed in both normal and neoplastic tissues. In normal tissues, nuclear dUTPase staining was observed exclusively in replicating cell types. This observation is in agreement with cell culture studies where expression of the nuclear isoform (DUT-N) is proliferation dependent In contrast, cytoplasmic expression of dUTPase does not correlate with proliferation status and was observed in tissues rich in mitochondria. Consistent with this observation, cell culture studies reveal that the mitochondrial isoform (DUT-M) is expressed constitutively, independent of cell cycle status. These data suggest that in normal tissues, nuclear staining with the DUT415 antibody represents the DUT-N isoform, whereas cytoplasmic staining represents the DUT-M isoform. In colon cancer tumor specimens, expression of dUTPase was shown to be highly variable in both amount and intracellular localization. Patterns of dUTPase protein expression observed included exclusive nuclear, exclusive cytoplasmic, and combined nuclear and cytoplasmic staining. Thus, immunohistochemical detection of dUTPase in colon cancers provides distinct intracellular phenotypes of expression that may be of significant prognostic value. To examine the association between dUTPase expression and response to 5-FU-based chemotherapy and overall survival, we initiated a retrospective study including tumor specimens from 20 patients who had received protracted infusion of 5-FU and leucovorin for treatment of metastatic colon cancer. Positive nuclear staining was found in 8 patients, whereas 12 lacked nuclear expression. Of the patients lacking nuclear dUTPase expression, 6 responded to 5-FU-based chemotherapy, 4 had stable disease, and 2 had progressive disease. Of the patients presenting positive nuclear dUTPase expression, 0 responded to chemotherapy, 1 had stable disease, and 7 had progressive disease (P = 0.005). The median survival for patients with tumors lacking nuclear staining was 8.5 months and 6.9 months for patients with tumors demonstrating positive nuclear dUTPase expression (P = 0.09). Time to progression was significantly longer for patients with tumors lacking nuclear staining (P = 0.017). Variable cytoplasmic dUTPase expression was observed in these tumors; however, there was no apparent association with clinical response or survival in this limited study. Nuclear dUTPase staining within these tumors was also associated with TS gene expression (P = 0.06). This study demonstrates that low intratumoral levels of nuclear dUTPase protein expression is associated with response to 5-FU-based chemotherapy, greater time to progression, and greater overall survival in colorectal cancer. Conversely, high levels of nuclear dUTPase protein expression predict for tumor resistance to chemotherapy, shorter time to progression, and shorter overall survival. This report represents the first clinical study implicating dUTPase overexpression as a mechanism of resistance to TS inhibitor-based chemotherapy.

Inflammation and infection imaging with a 99mTc-neutrophil elastase inhibitor in monkeys.

UNLABELLED: A radiolabeled human neutrophil elastase inhibitor (EPI-HNE-2) may represent an improved nuclear medicine imaging agent for inflammation and infection. This peptide displays rapid pharmacokinetics due to its low molecular weight and localizes specifically on neutrophil elastase released in inflammatory sites by activated neutrophils. METHODS: In this investigation, the peptide was radiolabeled with 99mTc using N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycline (NHS-MAG3) as a bifunctional chelator and was administered on 18 occasions to 5 rhesus monkeys with inflammation/infection. RESULTS: Plasma clearance was rapid, with liver and kidneys representing the major organs of accumulation. No evidence of toxicity, dosage effects, or circulating antiMAG3-EPI-HNE-2 antibodies was observed. Specificity of localization was established using radiolabeled bovine pancreatic trypsin inhibitor (a non-hNE-binding peptide of similar size) as a nonspecific negative control peptide and by predosing with unlabeled EPI-HNE-2 to block receptor sites before the administration of radiolabeled EPI-HNE-2. The ability of radiolabeled EPI-HNE-2 to image inflammation/infection was evaluated in 12 studies in monkeys receiving only radiolabeled EPI-HNE-2 and with lesions in the arm, shoulder, or lower back. Positive images were obtained in all studies, uptake was apparent almost immediately, and images were still positive 24 h later. As a positive control, animals also received nonspecific IgG antibody radiolabeled with 99mTc either directly or by NHS-MAG3. Compared with labeled antibody, plasma clearance of 99mTc was faster with labeled EPI-HNE-2 and accumulation in liver and heart was lower. Uptake of radioactivity in the inflammation was higher during the first hour with EPI-HNE-2 versus antibody but lower thereafter. CONCLUSION: When radiolabeled with 99mTc, EPI-HNE-2 localized specifically in inflammations in a monkey model and provided early images of diagnostic quality.

Fast nearest neighbor search of entropy-constrained vector quantization.

Entropy-constrained vector quantization (ECVQ) offers substantially improved image quality over vector quantization (VQ) at the cost of additional encoding complexity. We extend results in the literature for fast nearest neighbor search of VQ to ECVQ. We use a new, easily computed distance that successfully eliminates most codewords from consideration.

Phage display and pharmacogenomics.

Surface display of genetic diversity is a technology that can produce specific binding agents for almost any target molecule, and is especially well suited for making agents that bind specific proteins. Until now, pharmacogenomic studies have followed the response of cells to drugs and other agents by tracking the mRNAs that encode the proteins of interest, because it is relatively easy to produce nucleic acid ligands once the gene sequence is known. Nevertheless, in some cases, direct tracking of the proteins is preferred. Phage display of peptides and proteins (especially antibodies) is now able to provide the large number of specific binding agents needed to track proteins in pharmacogenomic studies.

Phase II/pharmacodynamic trial of dose-intensive, weekly parenteral hydroxyurea and fluorouracil administered with interferon alfa-2a in patients with refractory malignancies of the gastrointestinal tract.

PURPOSE: Combined depletion of pyrimidine and purine DNA precursors has resulted in therapeutic synergism in vitro. The aims of the current study were to test this strategy in patients with refractory tumors and to assess its effects on selected nucleotide pools. PATIENTS AND METHODS: A single-institution phase II trial was initiated in patients with advanced carcinomas of the stomach and pancreas. Patients had measurable disease and had no prior chemotherapy except adjuvant fluorouracil (5FU) or gemcitabine. 5FU was administered by CADD + pump at 2.6 g/m(2) intravenously by 24-hour infusion on days 1, 8, 15, 22, 29, and 36. Parenteral hydroxyurea (HU) was administered at 4.3 g/m(2) as a 24-hour infusion concurrently with 5FU. Interferon alfa-2a (IFN-alpha2a) was administered at 9 million units subcutaneously on days 1, 3, and 5 each week. No drug was administered in weeks 7 and 8. Pharmacodynamic studies were performed to assess drug effects on levels of deoxyuridine triphosphate (dUTP) and thymidine triphosphate (TTP) pools in peripheral-blood mononuclear cells (PBMCs) before and 6 hours after treatment using a highly sensitive DNA polymerase assay. RESULTS: There were 53 patients enrolled onto the study (gastric carcinoma, 31; pancreatic carcinoma, 22). The median age was 61 years, with 22% of patients > or = 70 years old. The predominant grade 3 to 4 toxicities were leukopenia (49%), granulocytopenia (55%), and thrombocytopenia (22%). Severe diarrhea occurred in 12%, mucositis in 0%, and vomiting in 10% of patients. Patients > or = 70 years had no greater incidence of toxicities. Among the 30 assessable patients with gastric carcinoma, there were two (7%) complete responders and 11 (37%) partial responders (median duration, 7 months). Among the 21 assessable patients with pancreatic carcinoma, there was one responder. Median survival among all patients with gastric carcinoma was 10 months and 13 months for patients with pancreatic carcinoma. Twenty-three patients had samples studied for levels of dUTP and TTP. There was no change in the levels of TTP before and after treatment. Furthermore, dUTP was detected in only five of 28 samples after treatment with no increase in the dUTP/TTP ratio. CONCLUSION: Combination therapy with high-dose, weekly infusional HU and 5FU with IFN-alpha2a modulation was well-tolerated with activity in gastric cancer. Patients > or = 70 years tolerated therapy as well as younger patients. This was the first study to correlate levels of TTP and dUTP after treatment with clinical outcome. In PBMCs used as a surrogate tissue, HU abrogated the 5FU-induced increase in dUTP levels without reversing the overall efficacy of the regimen.

Polypeptides from phage display. A superior source of in vivo imaging agents.

To decide whether experience teaches that small proteins and constrained peptides having high affinity for molecular targets can be engineered to have suitable pharmacokinetics for imaging. Phage display, a molecular diversity technology, allows selection of polypeptides having high affinity and specificity for almost any target. These polypeptides can be modified in ways that improve pharmacokinetics with acceptable impact on binding. Often, relatively few changes confers pharmacokinetics suitable for imaging on polypeptides selected for affinity and specificity to a target and for stability. It is likely that few variants of phage-selected proteins and constrained peptides will need to be tested to obtain a useful imaging agent.

Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.

We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.

Progressive transmission of images using MAP detection over channels with memory.

We propose a new maximum a posteriori (MAP) detector, without the need for explicit channel coding, to lessen the impact of communication channel errors on compressed image sources. The MAP detector exploits the spatial correlation in the compressed bitstream as well as the temporal memory in the channel to correct channel errors. We first present a technique for computing the residual redundancy inherent in a compressed grayscale image (compressed using VQ). The performance of the proposed MAP detector is compared to that of a memoryless MAP detector. We also investigate the dependence of the performance on memory characteristics of the Gilbert-Elliott channel as well as average channel error rate. Finally, we study the robustness of the proposed MAP detector's performance to estimation errors.

Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine.

UNLABELLED: A modified mercaptoacetyltriglycine (MAG3) chelator, which has acetyl S-protection and which is derivitized with N-hydroxysuccinimide (NHS) ester for conjugation, has been used to radiolabel four small (approximately 6- to 7-kDa) peptides, bovine pancreatic trypsin inhibitor, epidermal growth factor, human neutrophil elastase inhibitor and plasmin inhibitor, with 99mTc. METHODS: Each peptide was specifically labeled at the MAG3 chelation sites at ambient temperature and neutral pH. Specific activities of 100-150 mCi/mg were achieved at labeling efficiencies of about 50%, but specific activities of 3500 mCi/micromol could be attained. RESULTS: By a variety of assays, protein activity was unimpaired by the conjugation and labeling for two of the four peptides. The activities for plasmin of the plasmin inhibitor and bovine pancreatic trypsin inhibitor were reduced by conjugation, presumably because of a sensitive lysine residue in the structure of each of these two peptides. Multiple peaks were present in the high-performance liquid chromatography radiochromatograms, especially of human neutrophil elastase inhibitor; however, most peaks could be shown to be labeled active peptide. Stability during cysteine challenge at modest cysteine-to-peptide molar ratios and during incubation in serum was observed in each case. Large differences among the labeled peptides were apparent in the 3-hr biodistributions of 99mTc in normal mice. CONCLUSION: The use of NHS-S-acetyl-MAG3 may be a convenient method of radiolabeling peptides with 99mTc.

Measurement of deoxyuridine triphosphate and thymidine triphosphate in the extracts of thymidylate synthase-inhibited cells using a modified DNA polymerase assay.

New inhibitors of the enzyme thymidylate synthase (TS) are now reaching clinical application. Alteration of the dUTP: dTTP ratio may be critical to TS inhibition-induced tumor cell death. The DNA polymerase assay with modification was used to rapidly and sensitively measure dUTP, dTTP, and dUTP:dTTP ratios in cell extracts of HT29 human colon carcinoma cells treated with the specific TS inhibitor ZD1694 [N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-L-glutamic acid]. These results revealed an increase in the dUTP:dTTP ratio at 2 hr after a 2-hr exposure to ZD1694 at concentrations of 0.05 to 0.2 microM with significant normalization at 16 hr after a 2-hr exposure despite evidence of continued TS inhibition. This assay is highly sensitive and reproducible for levels of dUTP and is less labor intensive than traditional assays.

The human dUTPase gene encodes both nuclear and mitochondrial isoforms. Differential expression of the isoforms and characterization of a cDNA encoding the mitochondrial species.

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted Mr of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5' region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5' exons. This finding represents the first example in humans of alternative 5' exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.