Alterations in the ras oncogene and the p53 tumor suppressor gene in ultraviolet radiation-induced skin carcinogenesis

A rapid increase of the ultraviolet radiation (UVR)-related skin cancer incidence has attracted more and more public attention during the last few decades. Prevention and treatment of UVR-related skin cancer has become an important public health issue in the United States. Recent studies indicate that mutations in ras and/or p53 genes may be involved in UVR-induced skin tumor development but the precise molecular mechanism remains unclear. In this study, alterations of H-ras and p53 genes were investigated in different stages of carcinogenesis in a chronic UVR (solar simulator) exposure-induced Sencar mouse skin carcinogenesis model in order to clarify the role of the alterations of these genes during the skin carcinogenesis process and to further understand the mechanisms by which UVR causes skin cancer.^ Positive ras-p21 staining in cell membranes and cytosol were detected in 18/33 (55%) of squamous cell carcinomas (SCCs), but were not detected in UV-exposed skin, papillomas, or spindle cell tumors (SCTs). Positive staining of the malignant progression marker K13 was found in 17/33 (52%) of SCCs only. A significant positive correlation was observed between the K13 and the ras-p21 expression. Polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) analysis and gene sequencing analysis revealed three point mutations, one (codon 56) in UV-exposed non-tumor bearing skin and the other two (codons 21 and 13) in SCCs. No UV-specific mutation patterns were found.^ Positive p53 nuclear staining was found in 10/37 (27%) of SCCs and 12/24 (50%) of SCTs, but was not detected in normal skin or papillomas. PCR-based SSCP and sequencing analysis revealed eight point mutations in exons 5 and 6 (four in SCTs, two in SCCs, and two in UV-exposed skin) including six C-T or C-A transitions. Four of the mutations occurred at a dipyrimidine (CC) sequence. The pattern of the mutations indicated that the mutagenic lesions were induced by UVR.^ These results indicate that overexpression of ras-p21 in conjunction with aberrant expression of K13 occurred frequently in UVR-induced SCCs in Sencar mouse skin. The point mutation in the H-ras gene appeared to be a rare event in UVR skin carcinogenesis and may not be responsible for overexpression of ras-p21. UVR-induced P53 gene alteration is a frequent event in UVR-induced SCCs and later stage SCT tumors in Sencar mice skin, suggesting the p53 gene mutation plays an important role in skin tumor malignant progression. ^

Conidial germination of Botryosphaeria dothidea Mough.: Fr (Ces. & De Not.) and histological alterations on stems of pitahaya (Hylocereus undatus H.) (Haworth) Britton & Rose

Conidial germination of Botryosphaeria dothidea (anamorph: Fusicoccum) in sterile distilled water and 1% sterile dextrose solution was evaluated at 4, 6, 12, 24 and 36 h after incubation. Also, it was described the anatomical changes on pitahaya stems induced by this fungus, collected in the field and artificially inoculated in the laboratory. Conidial germination was less than 30% in water and it was improved when 1% dextrose was added to the water. In 1% dextrose solution the germination was 90% after 4h of incubation and 100% at 6 h. Pathogen germ tubes had entered through wounds and sometimes through stomata and hyphae colonized intra and intercellularly in the parenchyma-chlorenchyma tissues. On naturally and artificially diseased stems the main alterations were: destruction of cuticle, hyperplasia of epidermal and collenchymatous hypodermal cells and conform the advance of the pathogen a layer of lignified periderm was formed surrounding the damaged tissues; however, it couldn't stop the advance of the pathogen and the cells that surrounded the lesion suffered necrosis.

Brain rhythms alterations and their effect in functional networks: A simultaneous EEG/fMRI study in Fixation-off epilepsy

Fixation-off sensitivity (FOS) denotes the forms of EEG abnormalities, which are elicited by elimination of central vision or fixation. The phenomenon seems to depend on variables that modulate the alpha rhythm, however, the cerebral mechanisms underlying FOS remain unclear [1]. The scarce previous fMRI findings related to FOS have shown activation in extrastriate cortex [2] and also in frontal areas [3][4]. On the other hand, simultaneous EEG-fMRI technique has been used to assess the relationship between spontaneous power fluctuations of electrical rhythms and associated fMRI signal modulations. These studies have identified that lateral frontoparietal networks show a negative correlation with alpha band in healthy subjects. This neuroanatomical pattern is related to attentional processes and cognitive resources. Moreover, a sub-beta band (17-23 Hz) has been identified with posterior cingulate, temporoparietal junction and dorso-medial prefrontal cortex activations, which correspond to the DMN [5][6].

Alterations of cross-frequency coupling in MEG resting state recordings in Mild Cognitive Impairment patients

The default mode network (DMN) has received growing attention in recent years because it seems to be involved in the neuropathology of psychiatric and neurodegenerative disorders such as autism, schizophrenia and Alzheimer Disease. It has been defined as a task negative network, beca use the activity of all its brain regions is increased during the resting state and suspended during external or goal directed tasks.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30–42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Natural genetic exchange between Haemophilus and Neisseria: Intergeneric transfer of chromosomal genes between major human pathogens

Members of the bacterial families Haemophilus and Neisseria, important human pathogens that commonly colonize the nasopharynx, are naturally competent for DNA uptake from their environment. In each genus this process is discriminant in favor of its own and against foreign DNA through sequence specificity of DNA receptors. The Haemophilus DNA uptake apparatus binds a 29-bp oligonucleotide domain containing a highly conserved 9-bp core sequence, whereas the neisserial apparatus binds a 10-bp motif. Each motif (“uptake sequence”, US) is highly over-represented in the chromosome of the corresponding genus, particularly concentrated with core sequences in inverted pairs forming gene terminators. Two Haemophilus core USs were unexpectedly found forming the terminator of sodC in Neisseria meningitidis (meningococcus), and sequence analysis strongly suggests that this virulence gene, located next to IS1106, arose through horizontal transfer from Haemophilus. By using USs as search strings in a computer-based analysis of genome sequence, it was established that while USs of the “wrong” genus do not occur commonly in Neisseria or Haemophilus, where they do they are highly likely to flag domains of chromosomal DNA that have been transferred from Haemophilus. Three independent domains of Haemophilus-like DNA were found in the meningococcal chromosome, associated respectively with the virulence gene sodC, the bio gene cluster, and an unidentified orf. This report identifies intergenerically transferred DNA and its source in bacteria, and further identifies transformation with heterologous chromosomal DNA as a way of establishing potentially important chromosomal mosaicism in these pathogenic bacteria.

Loss of heterozygosity induced by a chromosomal double-strand break

The repair of chromosomal double-strand breaks (DSBs) is necessary for genomic integrity in all organisms. Genetic consequences of misrepair include chromosomal loss, deletion, and duplication resulting in loss of heterozygosity (LOH), a common finding in human solid tumors. Although work with radiation-sensitive cell lines suggests that mammalian cells primarily rejoin DSBs by nonhomologous mechanisms, alternative mechanisms that are implicated in chromosomal LOH, such as allelic recombination, may also occur. We have examined chromosomal DSB repair between homologs in a gene targeted mammalian cell line at the retinoblastoma (Rb) locus. We have found that allelic recombinational repair occurs in mammalian cells and is increased at least two orders of magnitude by the induction of a chromosomal DSB. One consequence of allelic recombination is LOH at the Rb locus. Some of the repair events also resulted in other types of genetic instability, including deletions and duplications. We speculate that mammalian cells may have developed efficient nonhomologous DSB repair processes to bypass allelic recombination and the potential for reduction to homozygosity.

Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug’s mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

The human XRCC9 gene corrects chromosomal instability and mutagen sensitivities in CHO UV40 cells

The Chinese hamster ovary (CHO) mutant UV40 cell line is hypersensitive to UV and ionizing radiation, simple alkylating agents, and DNA cross-linking agents. The mutant cells also have a high level of spontaneous chromosomal aberrations and 3-fold elevated sister chromatid exchange. We cloned and sequenced a human cDNA, designated XRCC9, that partially corrected the hypersensitivity of UV40 to mitomycin C, cisplatin, ethyl methanesulfonate, UV, and γ-radiation. The spontaneous chromosomal aberrations in XRCC9 cDNA transformants were almost fully corrected whereas sister chromatid exchanges were unchanged. The XRCC9 genomic sequence was cloned and mapped to chromosome 9p13. The translated XRCC9 sequence of 622 amino acids has no similarity with known proteins. The 2.5-kb XRCC9 mRNA seen in the parental cells was undetectable in UV40 cells. The mRNA levels in testis were up to 10-fold higher compared with other human tissues and up to 100-fold higher compared with other baboon tissues. XRCC9 is a candidate tumor suppressor gene that might operate in a postreplication repair or a cell cycle checkpoint function.

Macrophage inflammatory protein-2: Chromosomal regulation in rat small intestinal epithelial cells

Nonpathogenic, resident bacteria participate in the pathogenesis of inflammation in the small intestine, but the molecular messages produced by such bacteria are unknown. Inflammatory responses involve the recruitment of specific leukocyte subsets. We, therefore, hypothesized that butyrate, a normal bacterial metabolite, may modulate chemokine secretion by epithelial cells, by amplifying their response to proinflammatory signals. We studied the expression of the chemokine, macrophage inflammatory protein-2 (MIP-2) by the rat small intestinal epithelial cell line, IEC-6. Cells were stimulated with lipopolysaccharide or with interleukin 1β (IL-1β) and incubated with sodium butyrate. Acetylation of histones was examined in Triton X acetic acid–urea gels by PAGE. Unstimulated IEC-6 cells did not secrete MIP-2. However, lipopolysaccharide and IL-1β induced MIP-2 expression. Butyrate enhanced MIP-2 secretion both in lipopolysaccharide-stimulated and IL-1β-stimulated enterocytes; but butyrate alone did not induce MIP-2 expression. Butyrate increased the acetylation of histones extracted from the nuclei of IEC-6 cells. Furthermore, acetylation of histones (induced by trichostatin A, a specific inhibitor of histone deacetylase) enhanced MIP-2 expression by cells stimulated with IL-1β. In conclusion, trichostatin A reproduced the effects of butyrate on MIP-2 secretion. Butyrate, therefore, increases MIP-2 secretion in stimulated cells by increasing histone acetylation. We speculate that butyrate carries information from bacteria to epithelial cells. Epithelial cells transduce this signal through histone deacetylase, modulating the secretion of chemokines.

The ARS309 chromosomal replicator of Saccharomyces cerevisiae depends on an exceptional ARS consensus sequence

Autonomously replicating sequence (ARS) elements, which function as the cis-acting chromosomal replicators in the yeast Saccharomyces cerevisiae, depend upon an essential copy of the 11-bp ARS consensus sequence (ACS) for activity. Analysis of the chromosome III replicator ARS309 unexpectedly revealed that its essential ACS differs from the canonical ACS at two positions. One of the changes observed in ARS309 inactivates other ARS elements. This atypical ACS binds the origin recognition complex efficiently and is required for chromosomal replication origin activity. Comparison of the essential ACS of ARS309 with the essential regions of other ARS elements revealed an expanded 17-bp conserved sequence that efficiently predicts the essential core of ARS elements.

Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer. Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer. We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5′ and 3′ BRCA2 sequence information. Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.

Telomeres in the mouse have large inter-chromosomal variations in the number of T2AG3 repeats

The ultra-long telomeres that have been observed in mice are not in accordance with the concept that critical telomere shortening is related to aging and immortalization. Here, we have used quantitative fluorescence in situ hybridization to estimate (T2AG3)n lengths of individual telomeres in various mouse strains. Telomere lengths were very heterogeneous, but specific chromosomes of bone marrow cells and skin fibroblasts from individual mice had similar telomere lengths. We estimate that the shortest telomeres are around 10 kb in length, indicating that each mouse cell has a few telomeres with (T2AG3)n lengths within the range of human telomeres. These short telomeres may be critical in limiting the replicative potential of murine cells.

Specific cleavage of chromosomal and plasmid DNA strands in gram-positive and gram-negative bacteria can be detected with nucleotide resolution

A sensitive and precise in vitro technique for detecting DNA strand discontinuities produced in vivo has been developed. The procedure, a form of runoff DNA synthesis on molecules released from lysed bacterial cells, mapped precisely the position of cleavage of the plasmid pMV158 leading strand origin in Streptococcus pneumoniae and the site of strand scission, nic, at the transfer origins of F and the F-like plasmid R1 in Escherichia coli. When high frequency of recombination strains of E. coli were examined, DNA strand discontinuities at the nic positions of the chromosomally integrated fertility factors were also observed. Detection of DNA strand scission at the nic position of F DNA in the high frequency of recombination strains, as well as in the episomal factors, was dependent on sexual expression from the transmissable element, but was independent of mating. These results imply that not only the transfer origins of extrachromosomal F and F-like fertility factors, but also the origins of stably integrated copies of these plasmids, are subject to an equilibrium of cleavage and ligation in vivo in the absence of DNA transfer.

Chromosomal aberrations in PARP−/− mice: Genome stabilization in immortalized cells by reintroduction of poly(ADP-ribose) polymerase cDNA

Depletion of poly(ADP-ribose) polymerase (PARP) increases the frequency of recombination, gene amplification, sister chromatid exchanges, and micronuclei formation in cells exposed to genotoxic agents, implicating PARP in the maintenance of genomic stability. Flow cytometric analysis now has revealed an unstable tetraploid population in immortalized fibroblasts derived from PARP−/− mice. Comparative genomic hybridization detected partial chromosomal gains in 4C5-ter, 5F-ter, and 14A1-C1 in PARP−/−mice and immortalized PARP−/−fibroblasts. Neither the chromosomal gains nor the tetraploid population were apparent in PARP−/− cells stably transfected with PARP cDNA [PARP−/−(+PARP)], indicating negative selection of cells with these genetic aberrations after reintroduction of PARP cDNA. Although the tumor suppressor p53 was not detectable in PARP−/− cells, p53 expression was partially restored in PARP−/− (+PARP) cells. Loss of 14D3-ter that encompasses the tumor suppressor gene Rb-1 in PARP−/− mice was associated with a reduction in retinoblastoma(Rb) expression; increased expression of the oncogene Jun was correlated with a gain in 4C5-ter that harbors this oncogene. These results further implicate PARP in the maintenance of genomic stability and suggest that altered expression of p53, Rb, and Jun, as well as undoubtedly many other proteins may be a result of genomic instability associated with PARP deficiency.