Genetic linkage analysis identifies Pas1 as the common locus modulating lung tumorigenesis and acute inflammatory response in mice.

Selective breeding for the acute inflammatory response (AIR) generated two mouse lines characterized by maximum (AIRmax) and minimum (AIRmin) responses, explained by the additive effect of alleles differentially fixed in quantitative trait loci (QTLs). These mice also differ in their susceptibility to lung tumorigenesis, raising the possibility that the same loci are involved in the control of both phenotypes. To map the QTLs responsible for the different phenotypes, we carried out a genome-wide linkage analysis using single-nucleotide polymorphism arrays in a pedigree consisting of 802 mice, including 693 (AIRmax × AIRmin)F2 intercross mice treated with urethane and phenotyped for AIR and lung tumor multiplicity. We mapped five loci on chromosomes 4, 6, 7, 11 and 13 linked to AIR (logarithm of odds (LOD)=3.56, 3.52, 15.74, 7.74 and 3.34, respectively) and two loci linked to lung tumor multiplicity, on chromosomes 6 and 18 (LOD=12.18 and 4.69, respectively). The known pulmonary adenoma susceptibility 1 (Pas1) locus on chromosome 6 was the only locus linked to both phenotypes, suggesting that alleles of this locus were differentially fixed during breeding and selection of AIR mice. These results represent a step toward understanding the link between inflammation and cancer.

Osteoporosis detection by 3T diffusion tensor imaging and MRI spectroscopy in women older than 60 years.

Aim of this study was to evaluate the cancellous bone quality of postmenopausal women (age >60 years) by diffusion tensor imaging (DTI) using mean diffusivity (MD) and fractional anisotropy (FA) in combination with proton magnetic resonance spectroscopy (1H-MRS). 20 postmenopausal women older than 60 years were introduced to dual-energy X-ray absorptiometry (DXA) examination in femoral neck and to an MRI spectroscopy and DTI evaluation at 3T. We observed that fat fraction (FF) can discriminate healthy and osteoporotic patients. Water mean diffusivity (MD) and FA can discriminate the healthy group from osteopenic and osteoporotic group. MD/FF vs FA/FF graph extracted from the femoral neck identifies all healthy individuals, according to DXA results. DTI and spectroscopy protocol performed in the femoral neck could be highly sensitive and specific in identifying healthy subjects.

A major susceptibility locus to murine lung carcinogenesis maps on chromosome 6.

Lung tumours represent a major cause of death in humans, and although smoking represents the main pathogenetic factor, inheritance also plays a part. However, the identification of possible predisposing genetic factors is difficult, because of their low penetrance. We took advantage of murine strains that are genetically susceptible or resistant to lung tumour development, to map murine genes associated with susceptibility to lung carcinogenesis. An F2 population of urethan-treated A/J x C3H/He mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning mice was scored with 83 genetic markers. A chromosome 6 distal region, spanning 35 centiMorgans, contained a major lung tumour susceptibility locus. No other chromosomal region was significantly associated with lung tumour development.

Genetic mapping of a pulmonary adenoma resistance (Par1) in mouse.

Lung cancer, a major cause of death in the Western world, has a poor prognosis. So far, therapeutic strategies have had only a limited effect. Lung cancer risk is strongly associated with cigarette smoking and lung cancer pedigrees are rare. However, a possible polygenic nature of inherited predisposition to this cancer has been envisaged. Mouse inbred strains with inherited predisposition and resistance to lung cancer provide an important tool for the dissection of the genetics of this complex disease. The A/J strain carries the pulmonary adenoma susceptibility 1 (Pas1) locus and develops many lung tumours. We have mapped the M. spretus-derived locus that strongly resists the lung tumorigenesis in Pas1/+ mice. This locus, pulmonary adenoma resistance 1 (Par1) maps to mouse chromosome 11, near the Rara locus, with a lod score of 5.3. In Pas1/+ mice Par1 accounts for 23% of the phenotypic variance and 10 fold reduction in total tumour volume. These results provide evidence for a major resistance locus affecting the expression of an inherited predisposition to lung cancer.

A radiation hybrid transcript map of the mouse genome.

Expressed-sequence tag (EST) maps are an adjunct to sequence-based analytical methods of gene detection and localization for those species for which such data are available, and provide anchors for high-density homology and orthology mapping in species for which large-scale sequencing has yet to be done. Species for which radiation hybrid-based transcript maps have been established include human, rat, mouse, dog, cat and zebrafish. We have established a comprehensive first-generation-placement radiation hybrid map of the mouse consisting of 5,904 mapped markers (3,993 ESTs and 1,911 sequence-tagged sites (STSs)). The mapped ESTs, which often originate from small-EST clusters, are enriched for genes expressed during early mouse embryogenesis and are probably different from those localized in humans. We have confirmed by in situ hybridization that even singleton ESTs, which are usually not retained for mapping studies, may represent bona fide transcribed sequences. Our studies on mouse chromosomes 12 and 14 orthologous to human chromosome 14 show the power of our radiation hybrid map as a predictive tool for orthology mapping in humans.

Sensitivity to gains during risky decision-making differentiates chronic cocaine users from stimulant-naïve controls.

BACKGROUND: Chronic cocaine use has been consistently associated with decision-making impairments that contribute to the development and maintenance of drug-taking. However, the underlying cognitive processes of risk-seeking behaviours observed in chronic cocaine users (CU) have so far remained unclear. Here we therefore tested whether CU differ from stimulant-naïve controls in their sensitivity to gain, loss, and probability of loss information when making decisions under risk. METHOD: A sample of 96 participants (56 CU and 40 controls) performed the no-feedback version of the Columbia Card Task, designed to assess risk-taking in relation to gain, loss, and probability of loss information. Additionally, cognitive performance and impulsivity were determined. Current and recent substance use was objectively assessed by toxicological urine and hair analysis. RESULTS: Compared to controls, CU showed increased risk-seeking in unfavourable decision scenarios in which the loss probability was high and the returns were low, and a tendency for increased risk aversion in more favourable decision scenarios. In comparison to controls, CU were less sensitive to gain, but similarly sensitive to loss and probability of loss information. Further analysis revealed that individual differences in sensitivity to loss and probability of loss information were related to cognitive performance and impulsivity. CONCLUSION: Reduced sensitivity to gains in people with CU may contribute to their propensity for making risky decisions. While these alterations in gain sensitivity might directly relate to cocaine use per se, the individual psychopathological profile of CU might moderate sensitivity to loss information.

Specific gene expression profiles distinguish among functional allelic variants of the mouse Pthlh gene in transfected human cancer cells.

The mouse parathyroid hormone-like hormone (Pthlh) gene encodes three allelic variants characterized by amino acid substitutions that are associated with susceptibility (Pthlh(Pro)) or resistance (Pthlh(Thr) and Pthlh(SerAspTyr)) to two-stage skin carcinogenesis and to modulation of cell migration in vitro in transfected human cancer cells. cDNA microarray hybridization analysis of 8473 transcript clones revealed a similar gene expression profile for the Pthlh(Thr) and Pthlh(SerAspTyr) alleles but a distinct pattern for the Pthlh(Pro) allele, suggesting an association between a specific gene expression profile and biological function of the Pthlh alleles. Some of the genes modulated by the Pthlh alleles, e.g., ANXA1, CCL2, FN1 and TFF3, play a role in cell migration and may represent candidate targets for this Pthlh function. Our study demonstrates the potential usefulness of gene expression profiling of genetic variants for the functional characterization of candidate cancer modifier genes.

Identification of RASSF8 as a candidate lung tumor suppressor gene.

The RASSF8 gene, which maps close to the KRAS2 gene, contains a RAS-associated domain and encodes a protein that is evolutionarily conserved from fish to humans. Analysis of the RASSF8 transcript revealed a complex expression pattern of 5'-UTR mRNA isoforms in normal lung and in lung adenocarcinomas (ADCAs), with no apparent differences. However, RASSF8 gene transcript levels were approximately seven-fold-lower in lung ADCAs as compared to normal lung tissue. Expression of RASSF8 protein by transfected lung cancer cells led to inhibition of anchorage-independent growth in soft agar in A549 cells and reduction of clonogenic activity in NCI-H520 cells. These results raise the possibility protein encoded by RASSF8 is a novel tumor suppressor for lung cancer.

FEM analysis of RF breast ablation: multiprobe versus cool-tip electrode.

BACKGROUND: Radio-frequency ablation (RFA) has recently received much attention as an effective minimally invasive strategy for the local treatment of tumors. The purpose of this study was to evaluate the efficacy of single-needle cool-tip RF breast ablation in terms of temperature distribution and duration of the procedure as compared to multiprobe RF breast ablation. MATERIALS AND METHODS: Two different commercially available radiofrequency ablation needle electrodes were compared. Finite-element method (FEM) models were developed to simulate the thermoablation procedures. A series of ex vivo radiofrequency thermal lesions were induced to check the response of the FEM calculations. RESULTS: Data obtained from FEM models and from ex vivo procedures showed that cool-tip RF breast ablation assures better performances than multiprobe RF breast ablation in terms of temperature distribution and duration of the procedure. Histopathological analysis of the cool-tip RF thermoablated specimens showed successful induction of coagulation necrosis in the thermoablated specimens. CONCLUSION: Data obtained from FEM models and from ex vivo procedures suggest that the proposed cool-tip RF breast ablation may kill more tumor cells in vivo with a single application than the multiprobe RF breast ablation.

Quantitative analysis of genetic susceptibility to liver and lung carcinogenesis in mice.

One-week-old male and female mice of the A, BALB/c (C), and C3H/He (C3) strains and of the AC3 and CC3 F1 hybrids were treated with a single dose (300 mg/kg s.c.) of urethan and then kept without further treatment until 30 and 40 weeks (males) or 30 and 65 weeks (females). The degree of difference in susceptibility to hepatocarcinogenesis between the susceptible C3 and the resistant A and C mice, in the different age and sex groups, was 4-12-fold by the analysis of the number of nodules/cm3 (N/cm3), which represents an estimation of tumor frequency and was more than 400-fold as indicated by the percentage of liver volume occupied by nodules (V%), an estimation of tumor size. With regard to lung carcinogenesis, mice of the A strain proved 5-10- and 20-70-fold more susceptible than the C and the C3 mice, respectively, as indicated by the number of microscopically identified lung tumors/mouse (N), an estimation of tumor frequency. The lung tumor size, as estimated by the mean tumor volume (V), was similar in A and C mice but much higher in the A than in the C3 groups (13- approximately 1000-fold difference). The AC3 hybrid was highly susceptible to both liver and lung carcinogenesis. The CC3 hybrid was as susceptible to lung carcinogenesis as its C parent and had an intermediate susceptibility to hepatocarcinogenesis. Our results indicate that a major determinant in the genetic susceptibility to hepatocarcinogenesis, and perhaps to lung carcinogenesis too, is tumor growth, as evidenced by a much greater tumor size in genetically susceptible than resistant mice.

Factors influencing the expression of endogenous retrovirus-related sequences in the liver of B6C3 mice.

The expression of RNA transcripts from three families of endogenous retrovirus-related sequences was investigated during liver cell proliferation in B6C3 mice. Treatment with a single dose of the liver mitogen and promoter of mouse hepatocarcinogenesis 1, 4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), or with carbon tetrachloride (CCl4), induced liver cell proliferation at days 2 and 3 after treatment. Both of these treatments led to a marked increase in Moloney murine leukemia virus-related 6 kilobase RNAs, which were most abundant at day 1 after TCPOBOP treatment and at day 2 after CCl4. Intracisternal A particle-related 6 kilobase RNAs were markedly increased at days 1 and 2 after TCPOBOP and at days 1, 2, and 3 after CCl4. VL30-related transcripts were slightly decreased after TCPOBOP, but they were markedly increased at days 1 and 2 following CCl4. The livers of 15-day-old untreated mice contained about a 3-fold higher level of Moloney murine leukemia virus-related RNAs than adult liver. Intracisternal A particle-related 6-kilobase transcripts were present at 3-fold higher abundance in 7-day-old than in 15-day-old or adult liver. RNAs homologous to VL30 were detected at about the same levels in infant as well as adult livers. Inhibition of protein synthesis by the administration of cycloheximide to adult mice caused a marked increase in the amount of Moloney murine leukemia virus-, intracisternal A particle-, and VL30-related RNAs in the livers of the treated mice, suggesting the existence of labile proteins that normally regulate the abundance of these transcripts. We postulate that the amounts of these putative regulatory proteins vary during both normal development and carcinogenesis and also in response to specific agents that induce liver cell proliferation.

Loss of heterozygosity at chromosome 11q in lung adenocarcinoma: identification of three independent regions.

We examined the pattern of allelic loss in 76 adenocarcinomas of the lung using 14 highly informative microsatellite markers on the long arm of chromosome 11. Loss of heterozygosity was found in 48 of 76 tumors (63%). Three distinct regions of deletion were identified. The first region, the most centromeric, lies between markers D11S940 and CD3D: the second, delimited by markers D11S924 and D11S925, is estimated to be 4 Mb in length, and has never been previously described; a third, more telomeric region, the length of which is also estimated to be in the range of 4 Mb, is bracketed by loci D11S1345 and D11S1328. These findings suggest the presence of at least three tumor suppressor genes on the long arm of chromosome 11, and confirm the relevance of 11q22-24, a region frequently deleted in carcinomas of the breast, ovary, uterine, cervix, colon, and malignant melanoma in the pathogenesis of solid tumors. The characterization of minimal regions of loss could provide the basis for the identification and cloning of the critical genes.

Expression of retroviral sequences and oncogenes in murine hepatocellular tumors.

The expression of endogenous retroviral sequences and of three cellular oncogenes was examined in three hepatocellular adenomas and in four carcinomas induced in male C57BL/6JDp X C3Hf/Dp F1 (hereafter called B6C3F1) mice by a single dose of nitrosodiethylamine, in five carcinomas that arose spontaneously in male C3Hf mice, and in the livers of normal age-matched control mice. In all of these adenomas and carcinomas, there was increased expression of Moloney murine leukemia virus- and intracisternal A particle-related sequences. The retrovirus-like VL30 sequence was expressed at significant levels in the normal liver of these mice and increased expression of this sequence was found in only 4 of the 12 tumors examined. Expression of endogenous mouse mammary tumor virus-related sequences was not detected in the normal livers or in any of the liver tumors. With respect to cellular oncogenes, increased expression of c-myc was seen in all of the B6C3F1 tumors. Two of five normal liver samples and all of the tumors of the C3Hf mice displayed significant expression of c-myc. There was a slight increase in expression of c-Ha-ras in some of the tumors. Increased expression of c-fos was found in only 1 of the 12 tumors. Taken together, these studies indicate that both carcinogen-induced and spontaneous liver tumor formation in mice is associated with abnormalities in the expression of endogenous retrovirus-related DNA sequences and also specific cellular oncogenes.

Genetic mapping of lung cancer modifier loci specifically affecting tumor initiation and progression.

Mouse inbred strains with inherited predisposition and resistance to lung cancer provide a tool for the dissection of the complex genetics of this disease. In the present report, we have crossed the BALB/c with the SWR/J strain and performed whole-genome scanning for loci affecting lung tumor development in their F2 progeny. Both parental strains carry the pulmonary adenoma susceptibility 1 (Pas1) locus, a major locus affecting predisposition to lung cancer in mice. On distal chromosome 18 and on centromere of chromosome 6, we have mapped two pulmonary adenoma resistance loci (Par2 and Par4, respectively), which reduce lung tumor multiplicity strongly, up to 15-fold. Par2 and Par4, however, do not affect lung tumor size, which is instead controlled by an additional locus that we have mapped on the central region of chromosome 4. We designated this locus as "pulmonary adenoma progression 1" (Papg1), because it specifically modifies lung tumor size but not multiplicity. The present results, therefore, provide evidence for the existence of cancer modifier loci acting on specific stages of lung tumorigenesis.

Genetic evidence for an independent origin of multiple preneoplastic and neoplastic lung lesions.

Patients with a primary cancer in the lung or in the upper aerodigestive tract have an increased risk of developing synchronous or metachronous second primary lung tumors. This phenomenon has been related to the chronic exposure of the bronchial tree to carcinogens through a so-called "field cancerization" process. This study was designed to investigate at the somatic level the genetic basis of the field cancerization effect in patients having multiple simultaneous neoplastic and preneoplastic lesions of the lung. The pattern of specific genetic changes occurring with high frequency and in early stages of lung carcinogenesis including p53 mutations, deletions of chromosome 3p, and K-ras mutations, was investigated by immunocytochemical, cytogenetic, and molecular approaches in 11 synchronous lesions of five patients with multiple lung cancers. Different genetic lesions were observed in all of the pathological specimens analyzed from each patient. The pattern of these changes was different both in topographically distant or adjacent lesions and in tumors with the same histopathological diagnosis supporting their independent origin. The present data provide further evidence of the clinical relevance of the field cancerization process, and support the use of genetic markers in the differential diagnosis of recurrence or metastasis versus second primaries of the lung.

Chromosome mapping of murine susceptibility loci to liver carcinogenesis.

The validity of mouse liver tumors is controversial in the risk assessment of carcinogenicity of chemicals in humans, because mice used in carcinogenicity bioassays are genetically predisposed to liver tumors. The argument could be resolved once liver tumor susceptibility genes have been cloned and their role in liver tumor development elucidated. We performed a genetic linkage analysis to map murine liver tumor susceptibility genes, as a first step toward their identification. An F2 population of 87 urethane-treated male A/J x C3H/He mice was scored with 83 genetic markers. Three regions, localized on chromosomes 7, 8, and 12, were found to contain putative liver tumor susceptibility genes.

Population-based mapping of pulmonary adenoma susceptibility 1 locus.

Pulmonary adenoma susceptibility 1 (Pas1), the major locus affecting inherited predisposition to lung tumor development in mice, maps near the Kras2 gene. We previously reported a significant association between a KRAS2/RsaI polymorphism and the risk and prognosis of lung adenocarcinoma (ADCA) in the Italian population. In the present case-control study, we examined 269 lung ADCA patients, 121 squamous cell lung carcinoma patients, and 632 healthy individuals (general population controls) in the Japanese population with genetic markers spanning approximately 1200 kb in the KRAS2 region. Allele-specific oligonucleotide hybridization revealed the same KRAS2/RsaI polymorphism associated with risk and prognosis as in Italian lung ADCA patients; the polymorphism was significantly associated with clinical stage (P < 0.001) and survival rate (log rank = 0.0014), confirming the mapping of PAS1 and pointing to the role of this locus in human lung cancer.

The murine Fhit locus: isolation, characterization, and expression in normal and tumor cells.

The murine Fhit locus maps near the centromere nu proximal Ptprg locus on mouse chromosome 14. The cDNA sequence and structure are similar to those of the human gene, with exons 5-9 encoding the protein. The predominant mRNA in the tissues and cell lines tested was an alternatively spliced form missing exon 3. Most murine cell lines tested, including lines established from normal mouse embryos and tumors, expressed very low or undetectable levels of Fhit mRNA. Most normal mouse tissues expressed wild-type Fhit mRNA, whereas approximately 40% of murine lung carcinomas expressed wild-type and aberrant Fhit RT-PCR products that lacked various exons. Several tumorigenic mouse cell lines exhibited homozygous deletions of Fhit exons. We conclude that the murine Fhit gene, like its human counterpart, is a target of alterations involved in murine carcinogenesis.

Association between cigarette smoking and FHIT gene alterations in lung cancer.

Epidemiologic data have strongly indicated that cigarette smoking is linked to the development of lung cancer. However, little is known of the molecular targets of carcinogens contained in tobacco smoke. To identify genetic lesions characteristic of tobacco damage, we undertook a molecular analysis of microsatellite alterations within the FHIT gene and FRA3B, as well as at an independent locus on chromosome 10, D10S197, in lung tumors from heavy smokers and in tumors from never smokers. Loss of heterozygosity affecting at least one locus of the FHIT gene was observed in 41 of 51 tumors in the smokers group (80%) but in only 9 of 40 tumors in nonsmokers (22%). The comparison between the frequency of losses in FHIT in smokers and nonsmokers was statistically significant (P = 0.0001), whereas no difference in loss of heterozygosity rate was observed at D10S197 locus. These findings suggest that FHIT is a candidate molecular target of carcinogens contained in tobacco smoke.