Long-term safety of intramuscular gene transfer of non-viral FGF1 for peripheral artery disease.

Peripheral artery disease is a progressive disease. Primary ischemic leg symptoms are muscle fatigue, discomfort or pain during ambulation, known as intermittent claudication. The most severe manifestation of peripheral artery disease is critical limb ischemia (CLI). The long-term safety of gene therapy in peripheral artery disease remains unclear. This four center peripheral artery disease registry was designed to evaluate the long-term safety of the intramuscular non-viral fibroblast growth factor-1 (NV1FGF), a plasmid-based angiogenic gene for local expression of fibroblast growth factor-1 versus placebo in patients with peripheral artery disease who had been included in five different phase I and II trials. Here we report a 3-year follow-up in patients suffering from CLI or intermittent claudication. There were 93 evaluable patients, 72 of them in Fontaine stage IV (47 NV1FGF versus 25 placebo) and 21 patients in Fontaine stage IIb peripheral artery disease (15 NV1FGF versus 6 placebo). Safety parameters included rates of non-fatal myocardial infarction (MI), stroke, death, cancer, retinopathy and renal dysfunction. At 3 years, in 93 patients included this registry, there was no increase in retinopathy or renal dysfunction associated with delivery of this angiogenic factor. There was also no difference in the number of strokes, MI or deaths, respectively, for NV1FGF versus placebo. In the CLI group, new cancer occurred in two patients in the NV1FGF group. Conclusions that can be drawn from this relatively small patient group are limited because of the number of patients followed and can only be restricted to safety. Yet, data presented may be valuable concerning rates in cancer, retinopathy, MI or strokes following angiogenesis gene therapy in the absence of any long-term data in angiogenesis gene therapy. It may take several years until data from larger patient populations will become available.

BRCA1 and BRCA2 mutation status and cancer family history of Danish women affected with multifocal or bilateral breast cancer at a young age.

INTRODUCTION: A small fraction of breast cancer is the result of germline mutations in the BRCA1 and BRCA2 cancer susceptibility genes. Mutation carriers frequently have a positive family history of breast and ovarian cancer, are often diagnosed at a young age, and may have a higher incidence of double or multiple primary breast tumours than breast cancer patients in general. OBJECTIVES: To estimate the prevalence and spectrum of BRCA1 and BRCA2 mutations in young Danish patients affected with bilateral or multifocal breast cancer and to determine the relationship of mutation status to family history of cancer. SUBJECTS: From the files of the Danish Breast Cancer Cooperative Group (DBCG), we selected 119 breast cancer patients diagnosed before the age of 46 years with either bilateral (n=59) or multifocal (n=61) disease. METHODS: DNA from the subjects was screened for BRCA1 and BRCA2 mutations using single strand conformation analysis (SSCA) and the protein truncation test (PTT). Observed and expected cancer incidence in first degree relatives of the patients was estimated using data from the Danish Cancer Registry. RESULTS: Twenty four mutation carriers were identified (20%), of whom 13 had a BRCA1 mutation and 11 carried a BRCA2 mutation. Two mutations in BRCA1 were found repeatedly in the material and accounted for seven of the 24 (29%) mutation carriers. The mutation frequency was about equal in patients with bilateral (22%) and multifocal breast cancer (18%). The incidence of breast and ovarian cancer was greatly increased in first degree relatives of BRCA1 and BRCA2 mutation carriers, but to a much lesser degree in relatives of non-carriers. An increased risk of cancer was also noted in brothers of non-carriers. CONCLUSIONS: A relatively broad spectrum of germline mutations was observed in BRCA1 and BRCA2 and most of the mutations are present in other populations. Our results indicate that a diagnosis of bilateral and multifocal breast cancer is predictive of BRCA1 and BRCA2 mutation status, particularly when combined with information on the patients' age at diagnosis and family history of breast/ovarian cancer.

Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme.

The nucleoside triphosphatase (NTPase)/helicase associated with nonstructural protein 3 of West Nile (WN) virus was purified from cell culture medium harvested from virus-infected Vero cells. The purification procedure included sequential chromatography on Superdex-200 and Reactive Red 120 columns, followed by a concentration step on an Ultrogel hydroxyapatite column. The nature of the purified protein was confirmed by immunoblot analysis using a WN virus-positive antiserum, determination of its NH(2) terminus by microsequencing, and a binding assay with 5'-[(14)C]fluorosulfonylbenzoyladenosine. Under optimized reaction conditions the enzyme catalyzed the hydrolysis of ATP and the unwinding of the DNA duplex with k(cat) values of 133 and 5.5 x 10(-3) s(-1), respectively. Characterization of the NTPase activity of the WN virus enzyme revealed that optimum conditions with respect to the Mg(2+) requirement and the monovalent salt or polynucleotide response differed from those of other flavivirus NTPases. Initial kinetic studies demonstrated that the inhibition (or activation) of ATPase activity by ribavirin-5'-triphosphate is not directly related to changes in the helicase activity of the enzyme. Further analysis using guanine and O(6)-benzoylguanine derivatives revealed that the ATPase activity of WN virus NTPase/helicase may be modulated, i.e., increased or reduced, with no effect on the helicase activity of the enzyme. On the other hand the helicase activity could be modulated without changing the ATPase activity. Our observations show that the number of ATP hydrolysis events per unwinding cycle is not a constant value.

Inhibition of the helicase activity of HCV NTPase/helicase by 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5 '-triphosphate (ribavirin-TP).

In the presented study the ribavirin-TP--an established inhibitor of the NTPase activity of the superfamily NTPase/helicases II--was investigated as an inhibitor of the unwinding activity of the hepatitis C virus (HCV) NTPase/helicase. The kinetics of the reaction revealed that ribavirin-TP reduces the turnover number of the helicase reaction by a mechanism that does not correspond to that of the inhibition of the NTPase activity. Our results suggest that derivatives of ribavirin-TP with enhanced stability towards hydrolytic attack may be effective inhibitors of the enzyme.

ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy.

To enhance the inhibitory potential of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) vs hepatitis C virus (HCV) NTPase/helicase, ribavirin-5'-triphosphate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was prepared with the use of modified Yoshikawa-Ludwig-Mishra-Broom procedure (cf. Mishra & Broom, 1991, J. Chem. Soc., Chem. Commun, 1276-1277) involving phosphorylation of unprotected nucleoside. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50=40 microM) as compared to ribavirin (IC50 > 500 microM). Analysis of the inhibition type by means of graphical methods showed a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards nucleoside-5'-triphosphates (NTP) of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolyzed the ribavirin-TP to less potent products. Investigations on non- hydrolysable analogs of ribavirin-TP or ribavirin-5'-diphosphate (ribavirin-DP) are currently under way.

[Dissection and cytogenetic localization of the phenotypes of the 5p deletion syndrome].

OBJECTIVE: To classify the phenotypes of the short arm of chromosome 5 (5p) deletion syndrome and to localize their corresponding regions in the human genome. METHODS: A hundred and six patients, together with most of their parents and sibs, were analysed both cytogenetically and clinically. The dissection of the syndrome and cytogenetic localization of each phenotype was made by relating the clinical data to the breakpoints involved in the deletions. RESULTS: The 5p deletion syndrome was classified into 8 types based on the combination of the clinical data and high-resolution banding analysis. CONCLUSIONS: The dissection of the "5p deletion syndrome", a term with refined concept suggested by us, and the cytogenetic localization of each distinct phenotype will provide the clear cut criteria for clinical and cytogenetic diagnosis of the 5p-patients, as well as the candidate regions for positional cloning of the gene(s) repossible for the phenotypes.

Loss of the retinoblastoma protein-related p130 protein in small cell lung carcinoma.

The retinoblastoma gene family consists of the tumor suppressor protein pRB and its two relatives p107 and p130. These proteins have been implicated in the regulation of cell cycle progression, in part, through inactivation of members of the E2F transcription factor family. Overexpression of pRB, p107, or p130 leads to growth arrest in the G1 phase of the cell cycle, and this arrest is abolished by complex formation with the adenovirus E1A, human papilloma virus E7, or simian virus 40 T oncoproteins. Inactivation of pRB by gross structural alterations or point mutations in the RB-1 gene has been described in a variety of human tumors, including retinoblastomas, osteosarcomas, and small cell lung carcinomas. Despite the structural and functional similarity between pRB, p107, and p130, alterations in the latter two proteins have not been identified in human tumors. We have screened a panel of 17 small cell lung carcinoma cell lines for the presence of functional p107 and p130 by evaluating their ability to form complexes with E1A in vitro. In the GLC2 small cell lung carcinoma cells no p130 protein was detected. The loss of the p130 protein is the result of a single point mutation within a splice acceptor sequence in the GLC2 genomic DNA. This mutation eliminates exon 2, leading to an in-frame stop codon, and no detectable protein is produced. These data are, to our knowledge, the first to describe the loss of p130 as a consequence of a genetic alteration, suggesting that not only pRB but also the other members of the family may contribute to tumorigenesis, providing a rationale for the observation that the DNA tumor viruses selectively target all the members of the retinoblastoma protein family.