Polymorphisms in DNA repair genes in lung cancer patients living in a coal-mining region.

Air pollutants and ionizing radiation are well-known carcinogens involved in the pathogenesis of lung cancer, and residents of coal-mining regions are exposed routinely to these agents. Polymorphisms in DNA repair genes may be associated with an increased risk of malignant transformation. We investigated associations between the risk of lung cancer in residents of the coal-mining region and polymorphisms in the genes APEX1 (rs1130409), hOGG1 (rs1052133), XRCC1 (rs25489, rs25487), XRCC2 (rs3218536), XRCC3 (rs861539), ADPRT/PARP1 (rs1136410), XPD/ERCC2 (rs13181), XPG/ERCC5 (rs17655), XPC (rs2228001), ATM (rs1801516), and NBS1 (rs1805794). Three hundred and forty residents of the Kemerovo Region (a coal-mining region of western Siberia) were lung cancer patients exposed to air pollutants and ionizing radiation (case) and 335 were healthy donors (control). Genotyping was performed by real-time PCR and allele-specific PCR. We discovered that polymorphisms in the XPD gene in men [log-additive model: odds ratio (OR) = 1.64, 95% confidence interval (CI): 1.17-2.31], the ATM gene in women and nonsmokers (codominant model: OR = 0.11, 95% CI: 0.02-0.49 and OR = 0.25, 95% CI: 0.08-0.72, respectively), the APEX1 gene for smokers (recessive model: OR = 2.55, 95% CI: 1.34-4.85), and the NBS1 gene for those who work in the coal industry (overdominant model: OR = 0.40, 95% CI: 0.21-0.75) are associated with an increased risk of lung cancer. Using the multifactor dimensionality reduction method, we found a model of gene-gene interactions associated with the risk of lung cancer: NBS1 (rs1805794)-XRCC1 (rs25487)-hOGG1 (rs1052133)-XPG (rs17655). These results indicate an association between combinations of polymorphisms in the studied genes and the risk of lung cancer in residents of a coal-mining region.

New human single chain anti-idiotypic antibody against benzo[a]pyrene.

The nal¨ve library from the lymphocytes of healthy humans was screened by murine single-stranded idiotypic antibodies against benzo[a]pyrene (pSh). The phage clone which contained of anti-idiotypic antibody against benzo[a]pyrene, designated as A4, was chosen for further work because of highly specific to pSh. The available protein databases were searched. The A4 amino acid sequence was unique and 76% identical to a sequence in antibody against interferon g. The A4 protein was expressed in bacteria and purified by two different methods: His-tagged A4 and CBD-fusion A4. Both the A4 bound to pSh and also to the human single chain idiotypic antibody against the benzo[a]pyrene (T72) by ELISA. The Kd values of A4 for pSh and T72 were very close: 4.44 × 10-7 M and 5.71 × 10-7M, respectively. A4 was a competitor with benzo[a]pyrene for binding sites of both idiotypic pSh and T72 in competitive ELISA. Thus, A4 was a high affinity anti-idiotypic against benzo[a]pyrene which recognised pSh and T72 active sites.

Polymorphisms of GSTM1, GSTT1, GSTP1 genes and chromosomal aberrations in lung cancer patients.

PURPOSE: To study the potential links between genetic polymorphisms in the GSTT1, GSTM1, GSTP1 genes and the frequency of chromosomal aberrations (CAs) in lung cancer patients and healthy residents in Russian Federation. METHODS: 200 cells in well-spread metaphase with 46 chromosomes were examined for 353 newly diagnosed lung cancer patients (males) who received medical treatment in the Kemerovo Regional Oncology Center (Kemerovo, Russian Federation), and 300 healthy males from Kemerovo, Russian Federation. The polymorphisms of the GSTM1 del and GSTT1 del genes were analysed by multiplex PCR. Genotyping of the polymorphic variants in the GSTP1 (A313G, T341C) gene was performed using Real-time PCR with competing TaqMan probes complementary to the polymorphic DNA sites. The data analysis was performed using software STATISTICA 8.0 (StatSoft Inc., USA). RESULTS: We discovered that a GSTM1 del polymorphism increases the frequency of chromosomal damage in smoking patients with lung cancer, a general group of lung cancer patients, donors with non-small cell lung cancer and patients in the latest stages of the malignant process. The synergetic effects of occupational exposure and the malignant process can induce some modifications in the cytogenetic status in lung cancer patients harbouring the GSTM1 del polymorphism. CONCLUSIONS: CAs in peripheral blood lymphocytes can be used as biomarkers of the early biological effects of exposure to genotoxic carcinogens and may predict future cancer incidence in several epidemiologic studies. Genetic changes in genes encoding phase II detoxification enzymes are linked to decreases in the metabolic detoxification of environmentally derived genotoxic carcinogens.

Generation and Characterization of Human Single-Chain Antibodies Against Polycyclic Aromatic Hydrocarbons.

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed and relocated in the environment as a result of the incomplete combustion of organic matter. Many PAHs and their epoxides are highly toxic, mutagenic and/or carcinogenic to microorganisms as well as to higher systems including humans. BP is one of the most toxicologically active PAHs and is often used as a prototype for this entire class of contaminants. In order to select anti-BP antibodies, the conjugate of BP with BSA (BP-BSA) was used to screen naïve combinatorial phage library of human scFvs. Seven unique scFvs against BP-BSA were selected after three rounds of selection. Analysis of the genes encoding the scFvs subdivided them to gene families and subfamilies. Homology with the closest germline ranged from 80.21% to 97.57% for heavy chains and 88.89% to 98.57% for the light chains. Four of the seven scFv amino acid residues sequences without stop codons in frame were selected for proteomic analysis with each other. Four scFvs encoded unique non-related proteins with low-sequence identity among them. All CDRs and the boundaries in the CDR3 formation were carried out. Two of the scFvs (T68 and T72) with the highest binding capabilities to PAHs were expressed in E. coli and purified using a nickel resin. The KDs of T68 to BP-BSA, chrysene, pyrene, and benzo[a]anthracene were almost similar, approximately 10(-7 )M. The KDs of T72 to benzo[a]anthracene and chrysene were 9.42 × 10(-8 )M and 2.63 × 10(-7 )M, respectively. The computational models of T68 and T72 active centers were different.

Immunological disbalance in carcinogenesis.

It is postulated a conception of immunological disbalance between carcinogenesis inhibiting and stimulating antibodies (Ab). Inhibiting Ab prevent the carcinogens and estradiol but increase the progesterone penetration into the target cells. And vise versa do stimulating Ab. Inhibiting Ab could be blocked by corresponding antiidiotypic Ab. The processes of carcinogenesis initiation and promotion are intensified when stimulating Ab prevail over inhibiting ones.

Purification and characterization of mouse single-chain antibody against polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbons (PAH) such as benzo[a]pyren mainly induce lung cancer in humans. We characterized the mouse single chain antibody against benzo[a]pyren (pSh). pSh was expressed and purified as cellulose binding domain fusion (pSh-CBD). The pSh-CBD bound five different PAH with high affinity. The 18 amino acid linker connected pSh-CBD heavy and light chains provided correct protein folding. The KDs for pSh-CBD and polycyclic aromatic hydrocarbons were similar to KDs for monoclonal antibody, approximately 10(-8). Separately heavy and light chains of pSh-CBD did not interact with benzo[a]pyren. Previously defined eleven pSh-CBD aa involved to benzo[a]pyren binding were confirmed by mutagenesis.

Antisera to polycyclic aromatic hydrocarbons and their application on the detection of chemical carcinogens in blood sera of oncological patients.

AIM: To investigate the cross-reactivity of the hyperimmune antisera of animals (rabbits) and the sera of oncological patients to polycyclic aromatic hydrocarbons (PAHs) of a similar chemical structure. METHODS: Reactions of antibodies with haptens have been estimated by non-competitive and competitive ELISA with synthesized protein conjugates of PAHs as a coating antigene. RESULTS: All the model rabbit antisera have been stated to react with anthracene, chrysene, pyrene, benzo(a)pyrene and benz(a)anthracene irrespective of the hapten used to immunize animals. The cross-reactivity of serum antibodies to all five PAHs has been found in blood samples of oncological patients. CONCLUSION: It is recommended to use the conjugates of anthracene, chrysene and pyrene for detecting human antibodies to more dangerous environmental carcinogens--benzo(a)pyrene and benz(a)anthracene.

New approaches to immunoprevention and immunotherapy of neoplasms.

The problems in the creation of vaccines for the prevention and treatment of neoplasms and the perspectives of their application are discussed. The positive and negative properties of the conjugates of the chemical carcinogens with proteins, DNA and enterotoxins, as well as the properties of the monoclonal anti-idiotype antibodies to the carcinogens are being analyzed. We are describing here a new technology of the anti-carcinogen vaccine production that we have developed. We suggest using transgenic plants and bacteria carrying the Fv-genes of antibodies for the passive immunoprevention of cancer diseases. We assume that lymphoproliferative neoplasms mainly arise from the clones carrying membrane receptors for the carcinogens. Therefor, here it is proposed to use the xenogenic anticarcinogens antibodies for the active immunotherapy of leukemia and other lymphoproliferative neoplasms.

Synthesis of polycyclic aromatic hydrocarbon-protein conjugates for preparation and immunoassay of antibodies.

The method is described dealing with the synthesis of conjugates protein-polycyclic aromatic hydrocarbons (PAHs), highly soluble in water, stable without special stabilizers and containing the minimum quantity of cross-linked products. The reaction of protein with PAH containing an aldehyde group, has been carried out in an alkaline solution, and stabilization of the conjugate has been achieved by reduction with sodium borohydride in the presence of a compound blocking the formation of an insoluble polymeric fraction. The efficiency of synthesized conjugates for the induction and immunoassay of Abs to PAH for benzo[a]pyrene is shown.

Mechanisms of HLA DR Gene Selection in Early Ontogenesis.

Hypothesis explaining the cause of MHC gene frequency variation in two consecutive generations is presented. The four stages of MHC genes selection is postulated: gamete processing, reaction of fertilization, preimplatation and implatation embryos survival, postnatal selection. On the stage of gametogenesys different adaptivity of germ cells is connected with MHC alleles. On the stage of fertilization and implantation the main factor of selection is cell-to-cell polar interaction with the participation of MHC: spermatozoa and ovum; cleavaging cells of early embryo; cells of early embryo and endometrium. On the stage of development of implantation embryo the essential role play MHC-restricted immune mother-fetus interactions. Postnatal selection is connected with more or less resistance of children and adults to MHC-associated diseases.

Genetic Aspects of Immunopathology in Prenatal and Postnatal Period.

The development of immunopathology in prenatal and postnatal periods has been studied with special refer to immune interaction character in mother-fetus system and inherited HLA DR genes. The immune variant of customary recurrent abortion has been shown to be associated with the HLA DR2 and HLA DR6 genes, with poor immune recognition of fetus HLA antigens and absence of serum suppressive factors in women. The distorted immune interactions with HLA antigens in the mother-fetus system are transformed into the neonatal period as purulent-septic diseases and hemolytic disease of the newborns. The development of immunopathology, the maturation of main immunity parameters and postvaccinal immune response in the first-year-infants are associated with the inherited HLA DR genes.

Effect of Immune Interactions in Mother-Fetus System on Formation of Immune Response to Main Vaccinal Antigens.

Immune disturbances in the mother-fetus system are transformed to the postnatal period in the form of different kinds of immune pathology. Do these disturbances have an effect on the formation of postvaccinal immune response? A study was made in 81 children who were 14 months old. The immune response to diphtheria and measles antigen in those children was studied in the passive hemagglutination reaction. In order to detect immune disturbances in the mother-fetus system, their parents were tested using the methods: a mixed culture of lymphocytes (MLC) of the parents and determination of the blocking activity of the female serum (BAS) in MLC. The conducted study showed that there is a mutual relationship between the weak proliferative response in the MLC of the parents, combined with the absence of BAS, and low immune response to the diphtheria antigen. As regards measles vaccination, a close relationship between the strength of the immune response and immune reproductive disturbances has not been established. The obtained data may be used to predict the efficacy of alum precipitated DTP and DT vaccines.

Reaction of Immune System on Chemical Carcinogens.

Metabolism of carcinogens and their influence on the function of immunocompetent cells is quite well studied. However, well known view among immunologists and oncologists concerning the unambiguously inhibiting carcinogen action on immunity and failure of immunological surveillance is not quite correct. In 1937 H. Greech et al. were the first who reported that the immunization of animals with dibenzanthracene conjugates with proteins results in the appearance of antibodies to these carcinogens and inhibits the development of dibenzanthracene induced tumors. Antibodies and adducts carcinogen-DNA were found out in healthy individuals, subjected to strong carcinogen action. Using the conjugates carcinogen-protein, antibodies to carcinogens were revealed in cancer patients. However, antibodies were not detected in all the donors, having adducts; antibody binding to carcinogens was higher in cancer patients, than in healthy individuals; frequency detection of antibodies to carcinogens was considerably higher than in healthy donors. Anti-idiotypic antibodies to carcinogens have been appeared to be revealed by us in cancer patients (Glushkov et al., 1995). In a number of cases the appearance of anti-idiotypes (Ab2) was associated with absence of autoantibodies to carcinogens (Ab1). Individual isotypic differences in antibodygenesis seem to a larger degree to determine the proportion of organ and tissue resistance to carcinogen.

Analysis of Antibodies to Carcinogens and Oncoproteins Revealed by Onco-Immunological Screening.

The review highlights the role of environmental carcinogens (diolepoxides of benzpyrene (BP), benzanthracene (BA) and chrysene) for origin of cancer and analyses some recent data on methods to diagnose cancer diseases. To facilitate cancer diagnosing the method of specific Ab screening has been elaborated and investigated for being applied in clinical practice. It is suggested that high environmental concentrations of discussed chemicals may lead to significant accumulation of their carcinogenic metabolites in tissues, that may finally induces origin of cancer. The metabolites can form complexes with natural tissue macromolecules as RNA, DNA, serum blood proteins etc., termed as adducts, which are water soluble and finally excreted. Adducts with macromolecules can act as haptens and, as a rule, can trigger synthesis of specific Abs. It is proposed that elucidating of Abs specificity to particular carcinogens and dynamic of synthesis of Ig-Ab types may enable to diagnose particular stages of cancer, including those the very initial. Additionally, the Ab analysis may reveal which chemicals induced cancer. In connection with this, it seems perspective to elaborate and apply for cancer diagnosing a panel of mAbs to particular metabolites of the carcinogenic substances.