Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing.

Cancer suicide gene therapy affords the prospect of using the most optimal genes available because the source of the therapeutic gene is often irrelevant. Currently, there are numerous preclinical and clinical trials to develop tumor ablative therapies that use viral, yeast, or bacterial genes. One such gene, the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) is widely used as a suicide gene in combination with ganciclovir. In the study reported here, a restricted set of random sequences (semi-random) was introduced into the active site of HSV-1 TK, and the resulting variants were selected on the basis of their ability to confer increased ganciclovir or acyclovir sensitivity to Escherichia coli. Sequence analysis demonstrated that functional mutants contained three to five amino acid substitutions that are unique and novel combinations. On the basis of enzyme assay results, three mutants were identified for further analysis in vitro. These three mutants conferred substantial increased sensitivity to both ganciclovir and acyclovir when compared with IC50s of wild-type TK expressing rat C6 glioma cells. One mutant, SR39, was further evaluated in a xenograft tumor model in nude mice. Expression of SR39 in tumors was shown to prevent tumor growth at prodrug dosages that did not affect wild-type HSV-1 TK-expressing tumors. The use of any of these mutants as a suicide gene should provide a more effective and safer alternative to wild-type TK, because lower, less immunosuppressive doses of ganciclovir will be necessary for tumor ablation, and the use of acyclovir may now be possible.

Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein.

Sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaner population of South Africa, where a high incidence of the disorder occurs as a result of a founder effect. Homozygosity mapping in Afrikaner families along with analysis of historical recombinants localized sclerosteosis to an interval of approximately 2 cM between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Here we report two independent mutations in a novel gene, termed "SOST." Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The SOST gene encodes a protein that shares similarity with a class of cystine knot-containing factors including dan, cerberus, gremlin, prdc, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis, along with the data presented in this study, together suggest that the SOST gene encodes an important new regulator of bone homeostasis.

In vitro evaluation of mutant HSV-1 thymidine kinases for suicide gene therapy.

Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. While ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library of over a million mutant thymidine kinases, ten thymidine kinase variants were identified on the basis of activity towards ganciclovir and acyclovir (Black ME, Newcomb TG, Wilson H-MP and Loeb LA: Proc. Natl. Acad. Sci. U.S.A. 93: 3525-3529, 1996). Six mutants described here contain three to six amino acid changes and render mammalian cells more sensitive to acyclovir (ACV) including one that demonstrates an 8.5-fold reduction in IC50 compared to wild-type TK. These novel enzymes could provide benefit to ablative gene therapy by now making it feasible to use the relatively non-toxic acyclovir at nanomolar concentrations.

Enhancement of tumor ablation by a selected HSV-1 thymidine kinase mutant.

With the advent of gene therapy, herpes simplex virus type I (HSV-1) thymidine kinase (TK) has garnered much interest as a suicide gene for cancer ablation. As a means to improve the overall efficacy of the prodrug-gene activation approach, as well as to reduce ganciclovir-mediated toxicity, a large library of mutant thymidine kinases was generated and screened for the ability to enhance in vitro cell sensitivity to the prodrugs, ganciclovir (GCV) and acyclovir (ACV). Enzyme kinetics of one thymidine kinase mutant from this library that contains six amino acid substitutions at or near the active site reveals a distinct mechanism for providing enhanced prodrug-mediated killing in mammalian cells. In in vitro rat C6 cell prodrug sensitivity assays the TK mutant (mutant 30) achieves nanomolar IC50 values with GCV and ACV, in contrast to IC50values of 30 microM and >100 microM, respectively, for wild-type TK. In a mouse xenograft tumor model, growth of mutant 30 expressing tumors is restricted by ganciclovir at a dose at least 10- fold lower than one that impedes growth of wild-type TK-expressing tumors. Furthermore, in the presence of GCV a substantial bystander effect is observable when only 20% of the tumor cells express mutant 30 whereas no restriction in tumor growth is seen in tumors bearing the wild-type TK under the same conditions. The enhanced sensitization to prodrugs conferred by mutant 30 is apparently due to a 35-fold increase in thymidine Km which results in reduced competition between prodrug and thymidine at the active site. This provides mutant 30 a substantial kinetic advantage despite very high Kms for both ganciclovir and acyclovir. Molecular modeling of the mutations within the active site suggests that a tyrosine substitution at alanine 168 (A168) alters thymidine and prodrug interactions by causing catalytically important residues to move. The use of mutant 30 in place of the wild-type TK should provide a more effective gene therapy of cancer.

Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.

The live attenuated bacillus Calmette-Guérin (BCG) vaccine for the prevention of disease associated with Mycobacterium tuberculosis was derived from the closely related virulent tubercle bacillus, Mycobacterium bovis. Although the BCG vaccine has been one of the most widely used vaccines in the world for over 40 years, the genetic basis of BCG's attenuation has never been elucidated. We employed subtractive genomic hybridization to identify genetic differences between virulent M. bovis and M. tuberculosis and avirulent BCG. Three distinct genomic regions of difference (designated RD1 to RD3) were found to be deleted from BCG, and the precise junctions and DNA sequence of each deletion were determined. RD3, a 9.3-kb genomic segment present in virulent laboratory strains of M. bovis and M. tuberculosis, was absent from BCG and 84% of virulent clinical isolates. RD2, a 10.7-kb DNA segment containing a novel repetitive element and the previously identified mpt-64 gene, was conserved in all virulent laboratory and clinical tubercle bacilli tested and was deleted only from substrains derived from the original BCG Pasteur strain after 1925. Thus, the RD2 deletion occurred after the original derivation of BCG. RD1, a 9.5-kb DNA segment found to be deleted from all BCG substrains, was conserved in all virulent laboratory and clinical isolates of M. bovis and M. tuberculosis tested. The reintroduction of RD1 into BCG repressed the expression of at least 10 proteins and resulted in a protein expression profile almost identical to that of virulent M. bovis and M. tuberculosis, as determined by two-dimensional gel electrophoresis. These data indicate a role for RD1 in the regulation of multiple genetic loci, suggesting that the loss of virulence by BCG is due to a regulatory mutation. These findings may be applicable to the rational design of a new attenuated tuberculosis vaccine and the development of new diagnostic tests to distinguish BCG vaccination from tuberculosis infection.

Disparate responses to oxidative stress in saprophytic and pathogenic mycobacteria.

To persist in macrophages and in granulomatous caseous lesions, pathogenic mycobacteria must be equipped to withstand the action of toxic oxygen metabolites. In Gram-negative bacteria, the OxyR protein is a critical component of the oxidative stress response. OxyR is both a sensor of reactive oxygen species and a transcriptional activator, inducing expression of detoxifying enzymes such as catalase/hydroperoxidase and alkyl hydroperoxidase. We have characterized the responses of various mycobacteria to hydrogen peroxide both phenotypically and at the levels of gene and protein expression. Only the saprophytic Mycobacterium smegmatis induced a protective oxidative stress response analogous to the OxyR response of Gram-negative bacteria. Under similar conditions, the pathogenic mycobacteria exhibited a limited, nonprotective response, which in the case of Mycobacterium tuberculosis was restricted to induction of a single protein, KatG. We have also isolated DNA sequences homologous to oxyR and ahpC from M. tuberculosis and Mycobacterium avium. While the M. avium oxyR appears intact, the oxyR homologue of M. tuberculosis contains numerous deletions and frameshifts and is probably nonfunctional. Apparently the response of pathogenic mycobacteria to oxidative stress differs significantly from the inducible OxyR response of other bacteria.

Influence of meteorological events on obstetrical data in cattle and swine.

In a interdisciplinary scientific cooperation involving human and veterinarian obstetricians and meteorologists, an attempt was made to correlate the obstetrical data of cows and pigs with meteorologically defined weather events. During the observation period of 10 years the parturitions of cattle (n = 645) showed no significant statistical connection to the weather on day of delivery, the day before parturition or the day after. The only significant result in cattle was a shortening of the gestation period by 4.9 days in a group of cows that went into labour on, or after, the sixth day of a constant weather situation. Significant variations in the duration of gestation in swine (n = 786) were related to the weather on the day of delivery; central high-gradient anticyclonic spring, summer and autumn weather was linked to an extension of the pregnancy by nearly 1 day, while cyclonic central low-gradient weather during autumn was related to a shortening of pregnancy by approximately 1 day. The weather on the day before parturition was also correlated with the duration of gestation in swine. In this respect, low-gradient cyclonic autumn weather coincided with gestation periods that were reduced by 0.95 days, while central anticyclonic winter and spring weather coincided with extensions of gestation of 0.98 and 1.12 days, respectively. Compared with labour during low-gradient anticyclonic weather, the course of labour in pigs was significantly protracted by approximately 1.5 h during low-gradient cyclonic weather. Weather fronts on the day before, the day after or on the day of labour had no influence on the gestation and parturition data collected in either species.

[Is premature labor weather-dependent? Correlation of premature labor with meteorologic data]. / Besteht eine Wetterabhängigkeit der Frühgeburtlichkeit? Korrelation von Frühgeburten mit meteorologischen Ereignissen.

A preselected cluster of births (12351-4719 = 7632 = n) which was divided into 3 groups (delivery between 28-32 gestational weeks, 33-37 gestational weeks and > 37 gestational weeks) has been correlated with six major weather situations of the four meteorologically defined seasons. The correlation was made with the date of birth and with one day as well as two days before. The duration of pregnancy was longer by an average of 0.45 weeks (i.e. 3 days) when the major weather situation did not change for more than 8 days. The group with delivery 28-32 weeks is more sensitive to meteorological influences than the group with delivery 33-37 weeks. During cyclonic as well as during anticyclonic atmospheric drifts the groups 28-33 wks and 33-37 wks have been statistically over-represented. As far as the season is concerned over-representation is found in autumn. We conclude that pregnancies at risk of premature delivery should be followed up in short intervals during cyclonic and anticyclonic atmospheric drifts especially during the autumn season.

[Is there a gestosis weather? Correlation of EPH gestosis with meteorologic conditions]. / Gibt es ein Gestosewetter? Korrelation von EPH-Gestose mit meteorologischen Ereignissen.

A historical survey on the subject of weather and eclampsy is given in the introduction, at the beginning of which the names Smellie (1752), Boutteilloux (1816), Lachapelle (1925) and Zangemeister (1900) are mentioned. In the ten-year report the gestosis indices according to Goecke (n = 56,461) in the grouping 0/I-III/ = greater than IV are correlated with six general weather situations in each of the four seasons. From the chi 2 component test according to Krauth a relevant chi 2 limit of 8,95 is established. Only the following four out of altogether (6 x 4 =) 24 weather situations prove to be associated with lower (I-III) and higher (= greater than 4) indices of gestosis. 1. Anticyclonic situations with central high in winter with indices of gestosis I-III; 2. cyclonic flow-patterns in autumn with indices of gestosis I-III; 3. situations with central low in summer with indices of gestosis = greater than IV; 4. low gradient cyclonic situations in summer with fewer indices of gestosis. In spring the indices groups do not reach or exceed the limit of significance in any of the six weather situations. The four weather situations connected with gestosis are demonstrated by means of typical examples of infrared and visual satellite photos. Summing up, the different valence of scientific evidence and clinic relevance of the results is indicated.