Shigeto Hosoe

Methylation associated inactivation of RASSF1A from region 3p21.3 in lung, breast and ovarian tumours

Previously we analysed overlapping homozygous deletions in lung and breast tumours/tumour lines and defined a small region of 120 kb (part of LCTSGR1) at 3p21.3 that contained putative lung and breast cancer tumour suppressor gene(s) (TSG). Eight genes including RASSF1 were isolated from the minimal region. However, extensive mutation analysis in lung tumours and tumour lines revealed only rare inactivating mutations. Recently, de novo methylation at a CpG island associated with isoform A of RASSF1 (RASSF1A) was reported in lung tumours and tumour lines. To investigate RASSF1A as a candidate TSG for various cancers, we investigated: (a) RASSF1A methylation status in a large series of primary tumour and tumour lines; (b) chromosome 3p allele loss in lung tumours and (c) RASSF1 mutation analysis in breast tumours. RASSF1A promoter region CpG island methylation was detected in 72% of SCLC, 34% of NSCLC, 9% of breast, 10% of ovarian and 0% of primary cervical tumours and in 72% SCLC, 36% NSCLC, 80% of breast and 40% of ovarian tumour lines. In view of the lower frequency of RASSF1 methylation in primary breast cancers we proceeded to RASSF1 mutation analysis in 40 breast cancers. No mutations were detected, but six single nucleotide polymorphisms were identified. Twenty of 26 SCLC tumours with 3p21.3 allelic loss had RASSF1A methylation, while only six out of 22 NSCLC with 3p21.3 allele loss had RASSF1A methylation (P=0.0012), one out of five ovarian and none out of six cervical tumours with 3p21.3 loss had RASSF1A methylation. These results suggest that (a) RASSF1A inactivation by two hits (methylation and loss) is a critical step in SCLC tumourigenesis and (b) RASSF1A inactivation is of lesser importance in NSCLC, breast, ovarian and cervical cancers in which other genes within LCTSGR1 are likely to be implicated.

Localization of the von Hippel-Lindau disease gene to a small region of chromosome 3

We studied 25 families with von Hippel-Lindau disease (VHL) to locate VHL more precisely on chromosome 3. We found that VHL was linked to RAF1, confirming previous observations, and to two polymorphic DNA markers, D3S18 and D3S191. Multipoint linkage analysis indicated that the most likely location for VHL was in the interval between RAF1 and D3S18. D3S18 was located at 3p26. Genetic heterogeneity was not detected in this panel of von Hippel-Lindau disease families. The polymorphic markers RAF1, D3S18, and D3S191 should be useful in identifying asymptomatic gene carriers in VHL families and in guiding efforts at gene isolation.

Increased serum levels of basic fibroblast growth factor in lung cancer patients: relevance to response of therapy and prognosis

Angiogenesis is controlled by inhibitors and angiogenic factors. Among these, basic fibroblast growth factor (bFGF) is closely involved in cancer proliferation and has been related to progression and prognosis of various cancers, including lung cancer. To evaluate the role of bFGF, we measured serum levels of bFGF from healthy controls (Ctrl) and 106 patients with lung cancer, including 31 adenocarcinomas (AD), 29 squamous cell carcinomas (SQ), and 46 small cell carcinomas (SCLC), by enzyme-linked immunosorbent assays. Moreover, we evaluated the relationship between serum levels of bFGF and clinical outcome. Serum levels of bFGF in AD, SQ, SCLC, and Ctrl were 7.6 (0.5-32.5) (median (range)), 7.4 (0.5-36.7), 7.1 (0.5-34.8) and 3.0 (1.5-6.0) pg/ml, respectively (P<0.05). Serum bFGF levels did not differ between clinical stages in non-small cell lung cancer (NSCLC; AD+SQ). In SCLC, we found a significant difference in serum levels of bFGF between chemotherapy (and/or radiotherapy) responders (complete response+partial response) and non-responders (no change+progressive disease) (9.2 (0.6-34.8), 4.4 (0.5-17.4) pg/ml, respectively (P=0.018)), whereas there was no difference in NSCLC. Moreover, serum bFGF levels in SCLC patients had significant impact in prognosis by uni and multivariate analysis (P=0.014, 0.018, respectively). We concluded that bFGF has an important role in the prognosis of patients with SCLC.

Von Hippel-Lindau (VHL) disease: distinct phenotypes suggest more than one mutant allele at the VHL locus

SummaryAs part of an attempt to locate the von Hippel-Lindau locus (VHL) on chromosome 3, we evaluated 41 families with von Hippel-Lindau disease from the United States and Canada. One large family was identified whose disease phenotype was distinct from typical VHL. The most common disease manifestation was pheochromocytoma occuring in 57% (27/47) of affected family members. Few (4/47) affected family members had symptomatic spinal or cerebellar hemangioblastomas; no affected family member had renal cell carcinoma (0/47) or pancreatic cysts (0/24). Previously, genetic analysis demonstrated that the disease manifestations in this family were linked to RAF1 and D3S18, markers shown to be linked to typical VHL. These results suggest that there are mutant alleles at the VHL locus associated with distinct tissue specificities.

Phase II study of weekly irinotecan and carboplatin for refractory or relapsed small-cell lung cancer

We designed a phase II study of weekly irinotecan (CPT-11) and carboplatin for refractory or relapsed small cell lung cancer (SCLC) and assessed the response rate, survival, and toxicity. Twenty-nine patients with refractory or relapsed SCLC were entered onto the trial. The median time off chemotherapy was 3.5 months (range: 0.8-12.9). Patients were treated at 4-week intervals using CPT-11 (50 mg/m(2) intravenously on days 1, 8 and 15) plus carboplatin (AUC = 2 mg/ml min, intravenously on days 1, 8, 15). All patients were assessable for toxicity and survival; 28 patients were assessable for response. There were nine partial responses (PRs). Overall response rate was 31.0% (95% CI: 15.3-50.8%). The median time to progression was 3.5 months. Median survival time was 6.1 months. Major toxicity was myelosuppression. Grade 3 to 4 neutropenia and thrombocytopenia occurred in 52 and 21% of patients, respectively. Grade 3-4 diarrhea was observed in 7%. There was one treatment-related death due to febrile neutropenia and sepsis. This combination of CPT-11 and carboplatin seems to be active second-line regimen with acceptable toxicity against small cell lung cancer.

Detailed deletion mapping of the short arm of chromosome 3 in small cell and non-small cell carcinoma of the lung

We constructed a detailed deletion map of the short arm of chromosome 3 (3p) for 55 lung cancer cases by using 17 restriction fragment length polymorphism (RFLP) probes. Initially, we examined 40 small cell lung cancer (SCLC) cases and found three regions of deletion at 3p25-26, 3p21.3 and 3p14-cen, suggesting the possibility of at least three different tumor-suppressor genes on 3p. In order to obtain more detailed deletion area, and to compare the pattern of 3p deletion, we also examined 15 non-small cell lung cancer (NSCLC) cases. Compared to NSCLC cases, most of SCLC cases have widespread deletion on 3p, suggesting multiple tumor-suppressor genes on 3p may be inactivated in this type of cancer. In 3p21.3 area, minimum overlapping area of deletion lays between two probes which are close to each other. These data will be useful to isolate the putative tumor-suppressor genes located on the chromosome 3p.

Functional evidence for involvement of multiple putative tumor suppressor genes on the short arm of chromosome 3 in human oral squamous cell carcinogenesis

Cytogenetic and restriction fragment length polymorphism (RFLP) analyses have suggested that a putative tumor suppressor genes(s), which may play an important role in the development of human oral squamous cell carcinoma (SCC), is located on the short arm of chromosome 3 (3p). We previously reported that introducing in intact human chromosome 3 into three different oral SCC tumorigenic cell lines completely suppresses the tumorigenicity of each cell line with significant decrease in the in vitro growth rate and morphological changes. To map the tumor suppressor gene(s) on 3p, we have now examined the tumorigenicity of microcell hybrid clones containing various fragments derived from 3p that were introduced by microcell-mediated chromosome transfer. Sixteen hybrid clones were obtained from four successful experiments, and these clones were classified into two groups: 4 fully tumorigenic clones and 12 suppressed phenotype clones. Analyses of the 3p segments in the series of hybrid clones with the use of RFLP or microsatellite markers revealed that the 3p21.2-p21.3 or 3p25 regions or both were consistently retained in the 12 clones with suppressed phenotype but not in the 4 tumorigenic clones. The more proximal 3p13 region also was retained in three nontumorigenic clones. The overall results are fairly compatible with recent evidence that there are three discrete regions on 3p showing frequent allelic losses on oral SCC, and they directly provide functional evidence for the presence of tumor-suppressor genes for oral SCC in these regions. The possibility that three genes, FHIT, VHL, and T beta R-II, recently identified on 3p may be significantly involved in oral SCC development is also discussed.

Cloning and tissue expression of cDNAs from chromosome 5q21-22 which is frequently deleted in advanced lung cancer

Abstract Previously, we have reported that the inactivation of putative tumor-suppressor gene(s) on chromosome 5q21–22 may play an important role in the progression of lung cancer. Here, we describe the establishment of a yeast artificial chromosome (YAC) contig that spans 8–10 Mb at the 5q21–22 region. Six cosmid contigs have also been established in this YAC contig. About 35 exon-like fragments have been detected by exon-amplification, direct screening, cross-species hybridization, and searches of a database. Thus far, 14 cDNAs have been isolated, and two of them coincide with known genes, viz., cysteine dioxygenase I and geranylgeranyltransferase I. The other 12 cDNAs are considered to be novel genes. Two of these novel cDNA show partial homology to known genes, viz., semaphorin CD100 and the 28S rRNA gene. In addition, four known genes, including APC (adenomatous polyposis coli), MCC (mutated in colorectal cancer), proto-oncogene tyrosine kinase FER, and genomic imprinted gene U2AF1-RS1, have also been mapped in this contig. This large contig and expression map should prove crucial in the identification of susceptibility gene(s) related to the progression of lung cancer.

Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

SummaryA collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.

Induction of tumoricidal macrophages from bone marrow cells of normal mice or mice bearing a colony-stimulating-factor-producing tumor.

SummaryNonadherent cells of the bone marrow of C3H/HeN mice were incubated for 3 days with the culture supernatant of an L-929 cell line containing macrophage-colony-stimulating factor. Approximately, 70% of the cells became phagocytic, adherent to plastic dishes and positive for nonspecific esterase staining. The adherent cells exhibited a weak tumoricidal activity against MM48 syngeneic mammary carcinoma cells, and the cytotoxicity was strongly augmented by the addition of bacterial lipopolysaccharide to the cytotoxicity assay. The cytotoxicity induced by lipopolysaccharide was also shown to be mediated by Thy1.2− and asialo-GM1+ cells, and was abrogated by the addition of carrageenan. Macrophage-colony-stimulating-factor-producing (D66) and nonproducing (A23) variants were separated from the MM48 tumor line in in vitro culture following limiting dilution. There was no difference between these two variants in either the in vitro growth rate or the susceptibility to macrophage-mediated cytotoxicity. C3H/HeN mice inoculated i.p. with D66 survived longer than did those inoculated i.p. with A23. C3H/HeN mice bearing D66 or A23 as an ascitic form were given i.p. injections of Nocardia rubra cell wall skeleton (N-CWS). N-CWS significantly prolonged the survival period of mice bearing D66, whereas it exhibited no apparent antitumor effect on mice bearing A23. The increase in the cell number of D66 in the peritoneal cavity was significantly retarded, compared with that of A23. In contrast, the number of peritoneal macrophages increased more in D66-bearing mice than in A23-bearing mice. The increase in the peritoneal macrophage number was further augmented by an i.p. injection of N-CWS. Peritoneal macrophages of D66-bearing mice exhibited apparent tumoricidal activity against MM48 tumor cells in the presence of lipopolysaccharide, and the cytotoxicity was significantly augmented by i.p. injection of N-CWS. On the other hand, the responsiveness of peritoneal macrophages to lipopolysaccharide was found to be poor in A23-bearing mice and the tumoricidal activity was only weakly augmented by N-CWS. These results strongly suggest that M-CSF plays an important role not only in the maturation of macrophage progenitors but also in the induction and the accumulation of activated macrophages.