Kaiserslautern - Fachbereich Chemie
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Lung cancer, mainly caused by tobacco smoke, is the leading cause of cancer mortality. Large efforts in prevention and cessation have reduced smoking rates in the U.S. and other countries. Nevertheless, since 1990, rates have remained constant and it is believed that most of those currently smoking (~25%) are addicted to nicotine, and therefore are unable to stop smoking. An alternative strategy to reduce lung cancer mortality is the development of chemopreventive mixtures used to reduce cancer risk. Before entering clinical trails, it is crucial to know the efficacy, toxicity and the molecular mechanism by which the active compounds prevent carcinogenesis. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN) and benzo[a]pyrene (B[a]P) are among the most carcinogenic compounds in tobacco smoke. All have been widely used as model carcinogens and their tumorigenic activities are well established. It is believed that formation of DNA adducts is a crucial step in carcinogenesis. NNK and NNN form 4-hydroxy-1-(3-pyridyl)-1-butanone releasing and methylating adducts, while B[a]P forms B[a]P-tetraol-releasing adducts. Different isothiocyanates (ITCs) are able to prevent NNK-, NNN- or B[a]P-induced tumor formation, but relative little is know about the mechanism of these preventive effects. In this thesis, the influence of different ITCs on adduct formation from NNK plus B[a]P and NNN were evaluated. Using an A/J mouse lung tumor model, it was first shown that the formation of HPB-releasing, O6-mG and B[a]P-tetraol-releasing adducts were not affected when NNK and B[a]P were given individually or in combination, of by gavage. Using the same model, the effects of different mixtures of PEITC and BITC, given by gavage or in the diet, on DNA adduct formation were evaluated. Dietary treatment with phenethyl isothiocyanate (PEITC) or PEITC plus benzyl isothiocyanate (BITC) reduced levels of HPB-releasing adducts by 40*50%. This is consistent with a previously shown 40% inhibition of tumor multiplicity for the same treatment. In the gavage treatments with ITCs it seemed that PEITC reduced HPB-releasing DNA adducts, while levels of BITC counteracted these effects. Levels of O6-mG were minimally affected by any of the treatments. Levels of B[a]P-tetraol releasing adducts were reduced by gavaged PEITC Summary Page XII and BITC, 120 h after the last carcinogen treatment, while dietary treatment had no effects. We then extended our investigation to F-344 rats by using a similar ITC treatment protocol as in the mouse model. NNK was given in the drinking water and B[a]P in diet. Dietary PEITC reduced the formation of HPB-releasing globin and DNA adducts in lung but not in liver, while levels of B[a]P-tetraol-releasing adducts were unaffected. Additionally, the effects of PEITC, 3-phenlypropyl isothiocyanate, and their N-acetylcystein conjugates in diet on adducts from NNN in drinking water were evaluated in rat esophageal DNA and globin. Using a protocol known to inhibit NNNinduced esophageal tumorigenesis, the levels of HPB-releasing adduct levels were unaffected by the ITCs treatment. The observations that dietary PEITC inhibited the formation of HPB-releasing DNA adducts only in mice where the control levels were above 1 fmol/µg DNA and adduct levels in rat lung were reduced to levels seen in liver, lead to the conclusion that in mice and rats, there are at least two activation pathway of NNK. One is PEITC-sensitive and responsible for the high adduct levels in lung and presumably also for higher carcinogenicity of NNK in lung. The other is PEITC-insensitive and responsible for the remaining adduct levels and tumorigenicity. In conclusion, our results demonstrated that the preventive mechanism by which ITCs inhibit carcinogenesis is only in part due to inhibition of DNA adduct formation and that other mechanisms are involved. There is a large body of evidence indicating that induction of apoptosis may be a mechanism by which ITCs prevent tumor formation, but further studies are required.
The development of recombinant DNA techniques opened a new era for protein production both in scientific research and industrial application. However, the purification of recombinant proteins is very often quite difficult and inefficient. Therefore, we tried to employ novel techniques for the expression and purification of three pharmacologically interesting proteins: the plant toxin gelonin; a fusion protein of gelonin and the extracellular domain of the subunit of the acetylcholine receptor (gelonin-AchR) and human neurotrophin 3 (hNT3). Recombinant gelonin, acetylcholine receptor a subunit and their fusion product, gelonin-AchR were constructed and expressed. The gelonin gene, a 753 bp polynucleotide was chemically synthesized by Ya-Wei Shi et al. and was kindly provided to us. The gene was first inserted into the vector pUC118 yielding pUC-gel. It was subsequently transferred into pET28a and pET-gel was expressed in E. coli. The product, gelonin was soluble and was purified in two steps showing a homogeneous band corresponding to 28 kD on SDS-PAGE. The expression of the extracellular domain of the -subunit of AchR always led to insoluble aggregates and even upon coexpression with the chaperonin GroESL, very small and hardly reproducible amounts of soluble material were formed, only. Therefore, recombinant AchR- gelonin was cloned and expressed in the same host. The corresponding fusion protein, gelonin-AchR, again formed aggregates and it had to be solubilized in 6 M Gu-HCl for further purification and refolding. The final product, however, was recognized by several monoclonal antibodies directed against the extracellular domain of the -subunit of AchR as well as a polyclonal serum against gelonin. Expression and purification of recombinant hNT3 was achieved by the use of a protein self-splicing system. Based on the reported hNT3 DNA sequence, a 380 bp fragment corresponding to a 14 kD protein was amplified from genomal DNA of human whole blood by PCR. The DNA fragment was cloned into the pTXB1 vector, which contains a DNA fragment of intein and chintin binding domain (CBD). A further construct, pJLA-hNT3, is temperature-inducible. Both constructs expressed the target protein, hNT3-intein-CBD in E. coli by the induction with IPTG or temperature, however, as aggregates. After denaturation and renaturation, the soluble fusion protein was slowly loaded on an affinity column of chitin beads. A 14 kD hNT3 could be isolated after cleavage with DTT either at 4 °C or 25 °C for 48 h. Based on nerve fiber out-growth of the dorsal root ganglia of chicken embryos, both, hNT-3-intein-CBD and hNT3 itself exhibit almost the same biological activity.