The ras gene family and human carcinogenesis

It has been well established that specific alterations in members of the ras gene family, H-ras, K-ras and N-ras, can convert them into active oncogenes. These alterations are either point mutations occurring in either codon 12, 13 or 61 or, alternatively, a 5- to 50-fold amplification of the wild-type gene. Activated ras oncogenes have been found in a significant proportion of all tumors but the incidence varies considerably with the tumor type: it is relatively frequent (20-40%) in colorectal cancer and acute myeloid leukemia, but absent or present only rarely in, for example, breast tumors and stomach cancer. No correlation has been found, yet, between the presence of absence of an activated ras gene and the clinical or biological features of the malignancy. The activation of ras oncogenes is only one step in the multistep process of tumor formation. The presence of mutated ras genes in benign polyps of the colon indicates that activation can be an early event, possibly even the initiating event. However, it can also occur later in the course of carcinogenesis to initiate for instance the transition of a benign polyp of the colon into a malignant carcinoma or to convert a primary melanoma into a metastatic tumor. Apparently, the activation of ras genes is not an obligatory event but when it occurs it can contribute to both early and advanced stages of human carcinogenesis.     

NRAMP1 gene: structure,function, and human infectious diseases

The human infectious disease become of the great importance for Health Welfare. The infectious diseases mortality rate reaches one third of total mortality among 51 million patients died annually. The genetic factors seem to be most responsible for potency of human body to withstand to infections, caused by a variety of causative agents. The detection of the coincident factors and understanding the mechanisms of formation of susceptibility and resistance to infectious agents appeared to be important aspects for development of the new methods of prevention and treatment the infectious diseases. In inbred mice the natural resistance to infections, caused by Mycobacterium bovis, Mycobacterium avium, Mycobacterium lepraemurium, Leishmania donovani and Salmonella typhimurium is controlled by gene Nrampl (natural resistance associated macrophage protein gene). The gene codes for integral membrane protein, expressed by phagocytes. Protein is localized in the endosomal/lysosomal compartment of the silent macrophage, being recruited to the membrane of the phagosome containing particles under phagocytosis. This function is to transfer bivalent cations of metals from phagosome inward macrophage, that appears to affect negatively on destiny of microbes consumed. The human homologue of the Nrampl gene, denoted as NRAMP1, is situated on human chromosome 2q35. The gene Nrampl consists of 15 exones of different spread disparted by intrones of various sizes. Several polymorphous variants of the gene are described. The experimental presuppositions to more extensive investigation of the role of the gene NRAMPl in development of human pathology are pointed out.     

Epigenetic inactivation of the Ras-association domain family 1 (RASSF1A) gene and its function in human carcinogenesis

The Ras GTPases are a superfamily of molecular switches that regulate cellular proliferation and apoptosis in response to extra-cellular signals. The regulation of these pathways depends on the interaction of the GTPases with specific effectors. Recently, we have cloned and characterized a novel gene encoding a putative Ras effector: the Ras-association domain family 1 (RASSF1) gene. The RASSF1 gene is located in the chromosomal segment of 3p21.3. The high allelic loss in a variety of cancers suggested a crucial role of this region in tumorigenesis. At least two forms of RASSF1 are present in normal human cells. The RASSF1A isoform is highly epigenetically inactivated in lung, breast, ovarian, kidney, prostate, thyroid and several other carcinomas. Re-expression of RASSF1A reduced the growth of human cancer cells supporting a role for RASSF1 as a tumor suppressor gene. RASSF1A inactivation and K-ras activation are mutually exclusive events in the development of certain carcinomas. This observation could further pinpoint the function of RASSF1A as a negative effector of Ras in a pro-apoptotic signaling pathway. In malignant mesothelioma and gastric cancer RASSF1A methylation is associated with virus infection of SV40 and EBV, respectively, and suggests a causal relationship between viral infection and progressive RASSF1A methylation in carcinogenesis. Furthermore, a significant correlation between RASSF1A methylation and impaired lung cancer patient survival was reported, and RASSF1A silencing was correlated with several parameters of poor prognosis and advanced tumor stage (e.g. poor differentiation, aggressiveness, and invasion). Thus, RASSF1A methylation could serve as a useful marker for the prognosis of cancer patients and could become important in early detection of cancer.     

X-ray-induced oxidative stress: DNA damage and gene expression of HO-1, ERCC1 and OGG1 in mouse lung

Effects of X-ray induced oxidative stress in mouse lungs were studied in terms of DNA damage and expression of antioxidant defense and DNA repair genes. Lung samples were collected immediately after, and 3, 6, and 22 h after irradiation with 1, 3, 10 or 30 Gy X-rays of the thorax. The levels of strand breaks (SB), formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII) sensitive sites, detected by the comet assay, were increased dose-dependently immediately after irradiation, whereas 8-oxo-7,8-dihydro-2'-deoxyguanosine analyzed by HPLC-EC was unaltered, possibly due to a relatively high background level (2.5/10(6) dG in control tissue). Complete repair of SB was observed 3 h after irradiation, whereas the period required for repair of ENDOIII and FPG sensitive sites was longer. Determined by RT-PCR, the mRNA expression of heme oxygenase-1 (HO-1) was increased 40-fold 6 h after irradiation, whereas the expression of 8-oxoguanine glycosylase (OGG1) and ERCC1 were increased 2.5-fold 6 h after exposure, with saturation at the lowest dose. In conclusion, this study shows the feasibility of partial-body X-ray irradiation as an in vivo model for induction and repair of oxidative DNA damage, and expression of relevant DNA repair and antioxidant defense genes.     

The multi-step process of human skin carcinogenesis: a role for p53, cyclin D1, hTERT, p16, and TSP-1

As proposed by Hanahan and Weinberg (2000. Cell 100, 57-70) carcinogenesis requires crucial events such as (i) genomic instability, (ii) cell cycle deregulation, (iii) induction of a telomere length maintenance mechanism, and (iv) an angiogenic switch. By comparing the expression of p53, cyclin D1, p16, hTERT, and TSP-1 in spontaneously regressing keratoacanthoma (KA) as a paradigm of early neoplasia, with malignant invasive cutaneous squamous cell carcinoma (SCC) as a paradigm of advanced tumour development, we are now able to assign the changes in the expression of these proteins to specific stages and allocate them to defined roles in the multi-step process of skin carcinogenesis. We show that mutational inactivation of the p53 gene, and with that the onset of genomic instability is the earliest event. Individual p53-positive cells are already seen in "normal" skin, and 3/5 actinic keratoses (AKs), 5/22 KAs, and 13/23 SCCs contain p53-positive patches. Cell cycle deregulation was indicated by the overexpression of the cell cycle regulator cyclin D1, as well as by the loss of the cell cycle inhibitor p16. Interestingly, overexpression of cyclin D1 - observed in 80% of KAs and SCCs, respectively - showed a cell cycle-independent function in HaCaT cell transplants on nude mice. Cyclin D1 overexpression was associated with a massive inflammatory response, finally leading to tissue destruction. Loss of the cell cycle inhibitor p16, on the other hand, correlated with SCCs. Thus, it is tempting to suggest that overexpression of cyclin D1 is an early change that in addition to growth stimulation leads to an altered epithelial-mesenchymal interaction, while functional p16 is able to control this deregulated growth and needs to be eliminated for malignant progression. Another requirement for uncontrolled growth is the inhibition of telomere erosion by up-regulating telomerase activity. As measured by hTERT protein expression, all of the KAs and SCCs studied were positive, with a similar distribution of the protein in both groups and an expression pattern resembling that of normal epidermis. Thus, telomerase may not need to be increased significantly in skin carcinomas. Finally, we show that the angiogenesis inhibitor TSP-1 is strongly expressed in most KAs, and mainly by the tumour cells, while in SCCs the generally weak expression is restricted to the tumour-stroma. Furthermore, we provide evidence that the loss of a copy of chromosome 15 is responsible for reduced TSP-1 expression and thereby this aberration contributes to tumour vascularisation (i.e. the angiogenic switch) required for malignant growth.     

Subunit structure of human glucose 6-phosphate dehydrogenase and its genetic implication

Electrophoretic patterns of the subunits from the normal (B) and from the common A type variant of human glucose 6-phosphate dehydrogenase, following dissociation in the presence of 8m urea, were examined. A single protein band was found in both enzymes, indicating that the B and the A enzyme each consists of different subunits and do not have common subunits in their molecules. Since the A enzyme is a structural variant resulting from a single amino acid substitution, it can be concluded that the human G6PD consists of several identical subunits and that only one structural gene is involved in producing the enzyme. The findings of only a tyrosine residue as amino-terminal as well as a glycine residue as carboxy-terminal are in agreement with this conclusion.This study is supported by Public Health Service Grant GM 15253.     

Crystal structure of the TSP-1 type 1 repeats: a novel layered fold and its biological implication

Thrombospondin-1 (TSP-1) contains three type 1 repeats (TSRs), which mediate cell attachment, glycosaminoglycan binding, inhibition of angiogenesis, activation of TGFbeta, and inhibition of matrix metalloproteinases. The crystal structure of the TSRs reported in this article reveals a novel, antiparallel, three-stranded fold that consists of alternating stacked layers of tryptophan and arginine residues from respective strands, capped by disulfide bonds on each end. The front face of the TSR contains a right-handed spiral, positively charged groove that might be the recognition face, mediating interactions with various ligands. This is the first high-resolution crystal structure of a TSR domain that provides a prototypic architecture for structural and functional exploration of the diverse members of the TSR superfamily.     

Effect of single mutations in the OGG1 gene found in human tumors on the substrate specificity of the Ogg1 protein

We have investigated the effect of single amino acid substitutions of conserved arginines on the catalytic activities of the human Ogg1 protein (α-hOgg1-Ser³²⁶) (wild-type α-hOgg1). Mutant forms of hOgg1 with mutations Arg⁴⁶→Gln (α-hOgg1-Gln⁴⁶) and Arg¹⁵⁴→His (α-hOgg1-His¹⁵⁴) have previously been identified in human tumors. The mutant proteins α-hOgg1-Gln⁴⁶ and α-hOgg1-His¹⁵⁴ were expressed in Escherichia coli and purified to homogeneity. The substrate specificities of these proteins and wild-type α-hOgg1 were investigated using γ-irradiated DNA and the technique of gas chromatography/isotope-dilution mass spectrometry. All three enzymes excised 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 8-hydroxyguanine (8-OH-Gua) from γ-irradiated DNA containing a multiplicity of base lesions. Michaelis–Menten kinetics of excision were measured. Significant differences between excision kinetics of these three enzymes were observed. Excision of FapyGua and 8-OH-Gua by wild-type α-hOgg1 was greater than that by α-hOgg1-Gln⁴⁶ and α-hOgg1-His¹⁵⁴. The latter mutant protein was less active than the former. The diminished activity of the mutant proteins was more pronounced for 8-OH-Gua than for FapyGua. Cleavage assays were also performed using ³²P-labeled 34mer oligonucleotide duplexes containing a single 8-OH-Gua paired to each of the four DNA bases. The results obtained with the oligonucleotide containing the 8-OH-Gua/Cyt pair were in good agreement with those observed with γ-irradiated DNA. Wild-type α-hOgg1 and its mutants repaired the three mismatches less efficiently than the 8-OH-Gua/Cyt pair. The substitution of Arg¹⁵⁴, in addition to diminishing the activity on 8-OH-Gua, relaxes the selectivity found in the wild-type α-hOgg1 for the base opposite 8-OH-Gua. Taken together the results show that the mutant forms α-hOgg1-Gln⁴⁶ and α-hOgg1-His¹⁵⁴ found in human tumors are defective in their catalytic capacities.     

Expression of 15-lipoxygenase-1 in human nasal epithelium: its implication in mucociliary differentiation

15-lipoxygenase-1 (15-LO-1) is involved in the differentiation of human tracheobronchial epithelial cells. Here, we investigated the relation between 15-LO-1 expression and the differentiation of human nasal epithelium. In retinoic acid (RA)-sufficient culture media, 15-LO-1 expression in normal human nasal epithelial cell time-dependently increased, but its expression was undetectable in RA-deficient culture media. Moreover, in RA-deficient culture media, IL-4 at 1 ng/ml concentration time-dependently induced 15-LO-1 expression. In addition, MUC8 gene expression, a marker of mucociliary differentiation, was up-regulated by 15-LO-1, which was itself induced by IL-4. In murine nasal mucosa, the expression of leukocyte type-12-LO, a functional equivalent of 15-LO-1, reduced after postnatal day 7. Our findings suggest that 15-LO-1 is related to the differentiation of human nasal epithelium, and that it may mediate the mucociliary differentiation of human nasal epithelium.