Ken-ichi Kozaki

Polymerase Chain Reaction with Confronting Two-pair Primers for Polymorphism Genotyping

A novel PCR method using confronting two‐pair primers, named PCR‐CTPP, is introduced to detect a single nucleotide polymorphism (base X or Y). One primer for the X allele is set to include Xat the 3′end (antisense), where Xis the antisense of X, with the counterpart sense primer upstream. For the Y allele, a sense primer including Y at the 3′end is set, with the antisense primer downstream. One common band and one specific band for each allele are amplified, which allows genotyping directly by electrophoresis. This method is exemplified by application to the polymorphisms of beta‐adrenoceptor 2 and interleukin 1B. It is simpler than PCR‐RFLP (restriction fragment length polymorphism), which requires incubation with a restriction enzyme, and is suitable for genotyping in studies of genetic epidemiology involving hundreds of samples.

Isolation, purification, and characterization of a collagen-associated serpin, caspin, produced by murine colon adenocarcinoma cells.

A 45-kDa serpin secreted by a murine colon adenocarcinoma cell line, colon26, was isolated, purified, and characterized. It was found to bind specifically to type I collagen with high affinity and to type III collagen with lower affinity. Immunohistochemical studies of murine embryonic tissues showed a specific distribution of this collagen-associated serpin, named caspin, in relation to the formation of bone, cartilage, teeth, and basement membrane. The expression of caspin in high and low lung metastatic subclones of colon26 cell lines was inversely correlated with their metastatic capacity: low lung metastatic cells secreted higher amounts of caspin than their high lung metastatic counterparts. Caspin also demonstrated high homology with human pigment epithelium-derived factor/early population doubling level cDNA-1, which reportedly induces neuronal differentiation of human retinoblastoma cells and is expressed in association with G0 growth arrest. These findings suggest that caspin/pigment epithelium-derived factor/early population doubling level cDNA-1 is a novel factor that might play a crucial role in embryogenesis and tumor metastasis through binding to the extracellular matrix.

Detailed deletion mapping suggests the involvement of a tumor suppressor gene at 17p13.3, distal to p53, in the pathogenesis of lung cancers.

The short arm of chromosome 17 is one of the most frequently affected chromosomal regions in lung cancers, while there is solid evidence that the p53 gene at 17p13.1 is a target for frequent 17p deletions. In the present study, we re-evaluated 17p deletions in lung cancers by conducting a detailed analysis of the minimum deleted region(s) on 17p with reference to the p53 gene status in each 100 primary lung cancer cases. In addition to the p53 locus at 17p13.1, the presence of an independent, commonly deleted region(s) at 17p13.3 was identified. Furthermore, loss of heterozygosity (LOH) at 17p13.3 was shown to be even more frequent than that at 17p13.1 and it appeared to occur in the absence of p53 mutation and/or 17p13.1 deletion. These results suggest that in addition to the p53 gene at 17p13.1, an as yet unidentified tumor suppressor gene(s) residing at 17p13.3 might play a role in lung carcinogenesis possibly in an earlier phase than the p53 gene. This would warrant future studies to identify the putative tumor suppressor gene at 17p13.3 in order to gain a better understanding of the molecular pathogenesis of this fatal disease.

Significant up-regulation of a novel gene, CLCP1, in a highly metastatic lung cancer subline as well as in lung cancers in vivo

Most lung cancer patients are unfortunately uncurable and die because of widespread metastases, thus indicating the importance of identification of molecules with a crucial role in this process. Our previous expression profiling analysis of a highly metastatic lung cancer cell line, NCI-H460-LNM35, and its parental low metastatic line, NCI-H460-N15, revealed significant up-regulation of both known and unknown genes in LNM35. In this study, we describe the isolation and detailed characterizations of a novel gene, named CLCP1, which corresponds to one of such expression sequence tags with up-regulated expression in LNM35. The CLCP1 gene was found to encode a protein with 775 amino acids with structural similarities to, but distinct from neuropilins, cell surface receptors for VEGF165 and semaphorins. Notably, CLCP1 was shown to be up-regulated not only in LNM35 in association with its acquisition of metastatic phenotype during in vivo selection, but also in a significant fraction of lung cancers in vivo with high frequency in metastatic lesions, warranting future studies for a better understanding of the molecular mechanisms of lung cancer metastasis.

Protective Function of p27KIP1 against Apoptosis in Small Cell Lung Cancer Cells in Unfavorable Microenvironments

A previous study of ours unexpectedly found that in contrast to frequent reductions in non-small cell lung cancer, high expression of the p27(KIP1) cyclin-dependent kinase (CDK) inhibitor was retained in virtually all small cell lung cancers (SCLCs), suggesting the possibility of high expression of nonfunctional p27(KIP1) in this virulent tumor. The study presented here, however, shows that p27(KIP1) in SCLC biochemically functions as a CDK inhibitor, clearly showing induction apparently associated with G(1)/G(0) arrest and efficient binding to and inhibition of the cyclin E-CDK2 complex. Interestingly, induction of p27(KIP1) seems to confer on SCLC cells the ability to survive under culture conditions unfavorable for cell growth such as a lack of nutrients and hypoxia. Subsequent experiments manipulating p27(KIP1) levels by using a sense p27(KIP1) expression construct or an antisense oligonucleotide supported this notion. These observations suggest that high expression of p27(KIP1) in vivo may favor the survival of SCLC by preventing apoptosis in a microenvironment unfavorable for cell proliferation.

Development and Distribution of 2‐Amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]‐pyridine (PhIP)‐induced Aberrant Crypt Foci in the Rat Large Intestine

Aberrant crypt foci (ACF) are generally considered to be preneoplastic lesions for colon cancer. To assess their induction by 2‐amino‐1‐methyl‐6‐phenylimidazo[4,5‐b]pyridine (PhIP), a colon carcinogen, we performed a sequential study of ACF morphology and localization. F344 male rats were given PhIP, and methylene blue‐stained colon epithelium and isolated crypts were analyzed at weeks 12, 25, 50, and 75. Each crypt was classified into 2 groups, “single” with round bottoms and “bifurcating” displaying V‐shaped clefts (indicating proliferation). In combination with the number of crypts in an ACF, this classification was a good indicator for the generation of ACF in line with the fission mechanism of growth. Increasing numbers of crypts in ACF through weeks 12 to 75 and decreased percentages of ACF with bifurcating crypts at the late time points indicated that proliferation of crypts occurs predominantly during the early stages. The distribution pattern showed a significant shift (P < 0.000005) from the distal to the proximal part of the large intestine between weeks 25 and 50. Adenocarcinomas were first found to develop at week 50 in the ascending colon and cecum where bifurcating crypts were generally lacking at weeks 12 and 25. These data suggest the existence of (1) proliferating ACF which contains bifurcating crypt(s) and (2) quiescent or senescent ACF which consists of only single crypts.

Elevated expression of PDI family proteins during differentiation of mouse F9 teratocarcinoma cells

We investigated the expression of protein disulfide isomerase family proteins (PDI, ERp61, and ERp72) in mouse F9 teratocarcinoma cells during differentiation induced by treatment with retinoic acid and dibutyryl cAMP. Each member of this family was expressed at a constitutive level in undifferentiated F9 cells. During differentiation of F9 cells to parietal or visceral endodermal cells the protein level of all these enzymes increased, although the extent of this increase in both protein and mRNA levels varied among the enzymes. Certain proteins were found to be co‐immunoprecipitated with PDI, ERp61, and ERp72 in the presence of a chemical crosslinker. Type IV collagen was significantly coprecipitated with PDI whereas laminin was equally coprecipitated with the three proteins. Furthermore, 210 kDa protein characteristically coprecipitated with ERp72. Thus, the induction of PDI family proteins during the differentiation of F9 cells and their association with different proteins may implicate specific functions of each member of this family despite the common redox activity capable of catalyzing the disulfide bond formation. J. Cell. Biochem. 68:436–445, 1998.

Molecular Analysis of a Myc Antagonist, ROX/Mnt, at 17p13.3 in Human Lung Cancers

The chromosome region 17p13 is known to be frequently deleted in lung cancers. We recently showed the presence of an independent, commonly deleted region at 17p13.3, suggesting that in addition to p53 at 17p13.1 an as‐yet‐unidentified tumor suppressor gene may reside in this telomeric region. Interestingly, the chromosomal location of a recently isolated novel myc antagonist gene, termed ROX/Mnt, coincides exactly with the centromeric border of the commonly deleted region at 17p13.3 in lung cancers. In conjunction with the generally acknowledged roles of myc genes in the pathogenesis of lung cancers, these findings led us to investigate whether ROX/Mnt is altered in lung cancers. Despite an extensive search for alterations in 52 lung cancer specimens, somatic mutations of ROX/Mnt could not be identified. We conclude that ROX/Mnt itself is not a frequent target for 17p13.3 deletions in lung cancers and that further explorations are required to identify the putative tumor suppressor gene at 17p13.3.

Establishment and characterization of high- and low-lung-metastatic cell lines derived from murine colon adenocarcinoma 26 tumor line.

We established and characterized high‐ (LuM1) and low‐lung‐metastatic (NM11) cell lines derived from murine colon adenocarcinoma 26 tumor line. LuM1 cell line was established as a clonal cell line from a cultured cell mixture derived from a lung‐metastatic nodule after 7 sequential subcutaneous transplantations of lung‐metastatic tumors in the abdominal wall of BALB/c mice. NM11 cell line was established from a cultured cell mixture derived from a subcutaneous transplant of murine colon adenocarcinoma 26 tumor cells. LuM1 cells showed marked spontaneous lung metastases, but NM11 cells rarely did. High invasive potential of LuM1 cells was revealed by in vitro invasion assay using Matrigel reconstituted membranes. Rapid retraction was observed in monolayers of human umbilical vein endothelial cells and bovine aortic endothelial cells when LuM1 cells were added on the monolayers. Gelatin zymography and immunochemical examinations with monoclonal antibodies against gelatinase B (Mr 95,000 type IV collagenase) showed secretion of large amounts of the gelatinase by LuM1 cells.

Kinetics and collagenolytic role of eosinophils in chronic gastric ulcer in the rat

Abstractu2002An association between eosinophils and tissue damage has been observed in numerous disorders. However, few reports have addressed the role of infiltrating eosinophils in gastric ulcer healing. The aim of this study was to investigate the kinetics and role of eosinophils infiltrating experimental chronic gastric ulcers in the rat. We developed a monoclonal antibody against human matrix metalloproteinase 1 (MMP1) purified from conditioned culture medium of human skin fibroblasts. Acetic acid-induced gastric ulcers were resected from rats on days 1, 3, 5, 10, 20, 40, and 180 after the days of induction (day 0). Tissue specimens were immunostained with this antibody and examined with an electron microscope. Few eosinophils were observed in the granulation tissue until day 20. By days 40 and 180, MMP1-positive eosinophils had increased in the granulation tissue of open ulcers. Azan staining revealed dispersed collagen fibers around infiltrating eosinophils. In contrast, scars demonstrated few eosinophils in fibrous tissue on days 40 and 180. Eosinophils which express MMP1 infiltrate granulation tissue at the chronic stage of gastric ulceration. The results suggest that eosinophils may play a role in tissue remodeling and deterioration of ulceration.