Takeo Kakunaga

Transcriptional regulatory elements in the 5′ upstream and first intron regions of the human smooth muscle (aortic type) α-actin-encoding gene

Abstract We have determined the nucleotide (nt) sequence of 5.5 kb including the 5′ flanking, first untranslated exon and first intron regions of the human smooth muscle (SM) (aortic type) α-actin-(SMαA)-encoding gene. The promoter region and a part of the first intron show remarkably high sequence conservation with equivalent regions of the chicken gene, and contain multiple transcriptional regulatory elements. From transient chloramphenicol acetyltransferase gene (cat) expression assays m SM cells, a DNA fragment from nt − 123 to + 49 containing two CArG boxes showed strong positive promoter activity, whereas a far upstream region from nt − 253 to − 124 showed a negative effect. The conserved region in the first intron also contains the CArG box and showed an enhancer activity. Therefore, the human SMαA gene is controlled under positive and negative mechanisms.

A Simple and Useful Method for Simultaneous Screening of Elevated Levels of Expression of a Variety of Oncogenes in Malignant Cells

Abnormal expression of various oncogenes has been implicated in the development of many malignant tumors. Although RNA blotting methods have been used to measure abnormal expression, they involve the time‐consuming process of individually labeling the oncogene probes. To simplify this process we have attempted to develop a new method, termed simultaneous screening, which is based on the synthesis of radiolabeled cDNA corresponding to the mRNA population of malignant cells and on hybridization with various oncogene probes, immobilized on a membrane filter. This method circumvents the time‐consuming process of the prevailing RNA blotting methods and is also sensitive enough to detect accurately a five‐ to ten‐fold level of expression of rare mRNA (∼10 copies per cell). Overexpression of ten oncogenes was detected in a variety of malignant cells and mitogen‐stimulated cells with this method. These results suggest that our simultaneous screening method can be used to examine the overexpression of oncogenes.

An improved method of electroporation for introducing biologically active foreign genes into cultured mammalian cells

We have developed a modified, reproducible, and efficient method for introducing cloned genes into mammalian cells by using an electric field followed by treatment with sodium butyrate. Transfection frequencies with plasmid pSV2-neo, consisting of an antibiotic (G418) resistance gene and simian virus 40 (SV40) early promoter, by electroporation were higher than those by calcium phosphate DNA precipitation. Treatment with sodium butyrate following electroporation significantly increased the frequency of transfection in various types of cell lines and primary cultured cells including human skin fibroblasts. Treatment with sodium butyrate also increased the transient expression of the gene for chloramphenicol acetyltransferase (acetyl-CoA; chloramphenicol O3-acetyltransferase, CAT, EC 2.3.1.28) when the gene was introduced into BALB/c 3T3 cells by electroporation. Electroporation combined with sodium butyrate treatment is an improved method for stable and transient biochemical transformation of foreign genes in cultured mammalian cells.

Cross-linking of lipocortin I and enhancement of its Ca2+ sensitivity by tissue transglutaminase.

The stimulation of human epidermoid carcinoma A431 cells with the calcium ionophore A23187 resulted in the formation of high-molecular-weight lipocortins I, having apparent molecular weights of 75 kDa and 160 kDa as detected with specific anti-lipocortin I antibody. These immunoreactive proteins were identified to be covalently cross-linked multimers of lipocortin I, since essentially the same cross-linked multimers were observed when purified lipocortin I was incubated with tissue transglutaminase (TGase) in vitro. Classical amine substrates for TGase, such as dansylcadaverine and putrescine, were also incorporated stoichiometrically into lipocortin I. Cross-linking or amine incorporation was not observed with lipocortin II. Des 1-26 lipocortin I did not serve as a substrate for TGase, indicating that the N-terminal region of lipocortin I plays an important role in the formation of lipocortin I multimers. The cross-linking of lipocortin I by TGase resulted in a remarkable enhancement of calcium sensitivity for phospholipid binding; i.e., the free calcium concentration required for the cross-linked lipocortin I to attain 50% maximal binding to phosphatidylserine vesicles was as little as 3 microM, while that required for intact monomeric lipocortin I was 20 microM.

Isolation and characterization of three forms of 36-kDa Ca2+-dependent actin- and phospholipid-binding proteins from human placenta membrane.

We purified three forms of 36-kDa proteins, two monomeric 36-kDa proteins, which had pIs of 7.5 (36K-I) and 6.4 (36K-II), and one 36-kDa complex (36K-C) consisting of two subunits, 36-kDa (pI 7.5) and 12-kDa (pI 5.8), from human placenta membrane. The 36-kDa subunit of 36K-C was identical to 36K-I as judged by pI, cyanogen bromide peptide mapping and immunological cross-reactivity. The three proteins showed F-actin- and phosphatidylserine-binding abilities dependent on Ca2+ concentrations at millimolar and micromolar levels, respectively. They all had phospholipase A2 inhibitory activity. Only 36K-II was phosphorylated extensively at tyrosine residue in Ca2+- and EGF- dependent manners in the membrane fraction of A431 cells. 36K-I was the best substrate for src kinase, whereas 36K-II was the best for fps kinase. However, 36K-C was not phosphorylated by any kinases used here.

Altered expression of a third actin accompanying malignant progression in mouse B16 melanoma cells

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two‐dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found Ax actin (Mr = 43,000, pl = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high‐metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony‐forming ability following iv administration, increased with increase in the F number. The half life of Ax actin was much the same as that of β‐and γ‐actin and the different expressions of Ax actin between the low‐ (F=l) and high‐metastatic (F=10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of Ax actin. The Ax actin was incorporated into the cytoskeletal fraction with the same efficiency as β‐and γ‐actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in Ax expression. The actin stress fibers, observed staining with rhodamine‐conjugated phalloidin, were organized better in a low‐metastatic cell line (F=l) than in a high‐metastatic one (F = 10). These results suggest to us that depression of Ax actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta

Two monomeric 32‐kDa proteins, termed 32K‐I (pI 5.8) and 32K‐II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+‐chelator. Only 32K‐I was associated with PLA2‐inhibitory activity. CNBr peptide mapping indicated that 32K‐I was distinct from 32K‐II and two 36‐kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2‐inhibitory activity. 32K‐I bound to PS in a Ca2+‐dependent manner. 32K‐I was detected in many tissues except brain, cardiac and skeletal muscle.

A digital image processing system for quantitating dynamic morphology in cultured mammalian cells

An automated, video-driven system has been developed which can quantitate dynamic cell morphology in cultured mammalian cells. This system is based upon the Personal Image Analysis System and is assisted by a video-enhanced contrast microscopy with a computer-aided digital image processing unit and a time-lapse video technique. Various parameters for cell motility including locomotion (vectorial translation) and accompanying shape changes can be simultaneously analyzed. Here, we describe this system and demonstrate its application in Balb/c 3T3 cell culture. This system represents a new tool for exploring subtleties of mammalian cell behavior.

Only one type of benzo(a)pyrene-dna adduct is detected in transformable mouse cells.

Abstract When highly transformable BALB 3T3-A31 clone 1–13 cells are exposed to benzo(a)pyrene for various lengths of time, only one type of carcinogen-DNA adduct is detected. No minor adducts were found as reported in other cell systems. High pressure liquid chromatography identified this persistent adduct as 10-trans-7R-benzo(a)pyrene diolepoxide I-deoxyguanosine. Formation of benzo(a)pyrene-deoxyguanosine adduct, therefore, appears sufficient to initiate the transformation process induced by benzo(a)pyrene in 1–13 cells.

Establishment of cell lines derived from ataxia telangiectasia and xeroderma pigmentosum patients with high radiation sensitivity

Four human fibroblast cell lines, three of which were derived from a patient with ataxia telangiectasia and the other from a patient with xeroderma pigmentosum, were established after transfection with cloned SV40 DNA. These 4 cell lines showed some phenotypes characteristic of neoplastically transformed cells, and had a human karyotype with heteromorphisms identical to those of the parental fibroblasts. Their sensitivity to the cytotoxic effects of gamma-rays or ultraviolet irradiation was as high as those of their parental fibroblasts.