Growth Of Human Cell Lines Research Articles

Identification and characterization of the human N‐acetyltransferase complexes hNatB and hNatC.

Protein N‐alpha‐terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, three major complexes, NatA, NatB and NatC catalyse near all N‐alpha‐terminal acetylation, acetylating specific subsets of proteins. In humans, the NatA complex is the most extensively studied. We here present the human NatB and NatC complexes (hNatB and hNatC). They are conserved from yeast with respect to subunit composition, substrate specificity and ribosome binding. Knockdown of hNatB subunits disrupts normal cell cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL‐62. Knockdown of hNatC subunits results in p53‐dependent cell death and reduced growth in human cell lines. Taken together, these studies emphasize the biological importance of N‐alpha‐terminal acetylation.

Targeting PIM Kinases Impairs Survival of Hematopoietic Cells Transformed by Kinase Inhibitor–Sensitive and Kinase Inhibitor–Resistant Forms of Fms-Like Tyrosine Kinase 3 and BCR/ABL

Previous studies have shown that activation of the signal transducer and activator of transcription 5 (STAT5) plays an essential role in leukemogenesis mediated through constitutive activated protein tyrosine kinases (PTK). Because PIM-1 is a STAT5 target gene, we analyzed the role of the family of PIM serine/threonine kinases (PIM-1 to PIM-3) in PTK-mediated transformation of hematopoietic cells. Ba/F3 cells transformed to growth factor independence by various oncogenic PTKs (TEL/JAK2, TEL/TRKC, TEL/ABL, BCR/ABL, FLT3-ITD, and H4/PDGFbetaR) show abundant expression of PIM-1 and PIM-2. Suppression of PIM-1 activity had a negligible effect on transformation. In contrast, expression of kinase-dead PIM-2 mutant (PIM-2KD) led to a rapid decline of survival in Ba/F3 cells transformed by FLT3-ITD but not by other oncogenic PTKs tested. Coexpression of PIM-1KD and PIM-2KD abrogated growth factor-independent growth of Ba/F3 transformed by several PTKs, including BCR/ABL. Targeted down-regulation of PIM-2 by RNA interference (RNAi) selectively abrogated survival of Ba/F3 cells transformed by various Fms-like tyrosine kinase 3 (FLT3)-activating mutants [internal tandem duplication (ITD) and kinase domain] and attenuated growth of human cell lines containing FLT3 mutations. Interestingly, cells transformed by FLT3 and BCR/ABL mutations that confer resistance to small-molecule tyrosine kinase inhibitors were still sensitive to knockdown of PIM-2, or PIM-1 and PIM-2 by RNAi. Our observations indicate that combined inactivation of PIM-1 and PIM-2 interferes with oncogenic PTKs and suggest that PIMs are alternative therapeutic targets in PTK-mediated leukemia. Targeting the PIM kinase family could provide a new avenue to overcome resistance against small-molecule tyrosine kinase inhibitors.

Alkaloids from Menispermum dauricum

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Alkaloids from Menispermum dauricum

The alkaloids, dechloroacutumidine and 1-epidechloroacutumine, together with three known alkaloids, acutumidine, acutumine, and dechloroacutumine, were isolated from the rhizomes of Menispermum dauricum and their structures established by spectral and chemical methods. The cytotoxicity of each compound against the growth of human cell lines was studied, and acutumine selectively inhibited T-cell growth.

Antiproliferative activity and phototoxicity of some methyl derivatives of 5-methoxypsoralen and 5-methoxyangelicin.

The in vitro antiproliferative activity and in vivo phototoxicity of some methyl derivatives of 5-methoxypsoralen and 5-methoxyangelicin, i.e. 4,4'-dimethyl-5-methoxyangelicin (compound I), 3,4'-dimethyl-5-methoxyangelicin (compound II), 4,4'-dimethyl-5-methoxypsoralen (compound III); and 3,4'-dimethyl-5-methoxypsoralen (compound IV), have been investigated. The effects of the compounds were evaluated in vitro on HL60 and A431 cells, using 5-methoxypsoralen as the reference compound. In both cell lines compound I, II and III showed better antiproliferative activity than compound IV and 5-methoxypsoralen. Scanning electron microscopy revealed that all the compounds induced the formation of blebs and blisters on a A431 cell surface. Significant variations in the nuclear area strictly related to the toxicity of the compounds have been shown in both cell lines. Skin irritancy in vivo was evaluated by mean of histopathological responses on guinea-pig skin. For each compound a damage index was determined by morphometrical analysis of empty spaces in the epidermis. Histopathology revealed skin phototoxicity of compounds which lacked erythemogenic activity by visual scoring. By coupling cytotoxicity data in vitro to skin sensitization ones in vivo, compound I proved a promising candidate for use in clinical trials since due to a high inhibitory effect on the growth of human cell lines coupled to low skin phototoxicity.

Treatment of severe combined immunodeficiency mice with anti-murine granulocyte monoclonal antibody improves human leukocyte xenotransplantation.

The residual resistance of severe combined immunodeficiency (SCID) mice to human graft is the main factor in conditioning both the extent of human cell reconstitution and the xenograft-to-xenograft variability. We have recently shown that an early and massive murine granulocyte recruitment is the main event in the SCID mouse reaction to the human graft. Here, we evaluate the importance of mouse granulocytes in the restriction of human cell engraftment in SCID mice. We injected SCID mice with a monoclonal antibody to murine granulocytes. Injection of this antibody resulted in a marked depletion of polymorphonuclear cells in the hematopoietic organs of SCID mice. This depletion was associated with a significant increase in both the growth of human cell lines of different hematopoietic origin and the engraftment of human peripheral blood leukocytes. Moreover, the abolishment of the early granulocyte reaction markedly reduced the xenograft-to-xenograft variation, a major shortcoming of these xenochimeric models. These results provide new insights into the control of the natural immune response of SCID mice against human graft. Furthermore, treatments aimed at controlling the acute inflammatory reaction of SCID mouse-to-human cell transplantation can be considered useful experimental approaches for increasing the xenograft-to-xenograft reproducibility.

野菜抽出物のヒト培養細胞株の増殖に対する作用のＷＳＴ‐１アッセイによる検討

8種類のヒト培養細胞株を用い,7種類の野菜抽出物添加が細胞増殖に与える影響をWST-1アッセイにより検討した.ピーマン抽出物はほとんどの細胞に対して強い増殖抑制作用を示した.また,キャベツ抽出物はK562に対して強い増殖促進作用を示した.WST-1アッセイは多数の試料をスクリーニングするのに有用な測定法であった.

Antigen forks: bispecific reagents that inhibit cell growth by binding selected pairs of tumor antigens.

Bispecific antibodies of a new category, termed "antigen forks", were constructed by crosslinking antibodies that recognized pairs of distinct tumor cell surface antigens. At concentrations of 1-100 nM, several such forks inhibited the growth of human tumor cell lines bearing both relevant antigens. The same cells were not inhibited by unconjugated component antibodies, and the active conjugates did not inhibit the growth of human cell lines that expressed lower levels of relevant antigens. The three most active antigen forks all contained monoclonal antibody 454A12, which recognizes human transferrin receptor. This antibody was conjugated respectively to antibodies 113F1 (against a tumor-associated glycoprotein complex), 317G5 (against a 42-kDa tumor-associated glycoprotein), or 520C9 (against the c-erbB-2 protooncogene product). The 317G5-454A12 fork strongly inhibited the HT-29 and SW948 human colorectal cancer cell lines, while the 113F1-454A12 and 520C9-454A12 forks strongly inhibited the SK-BR-3 human breast cancer cell line and the 113F1-454A12 fork was also effective against SW948. By designing forks against antigens of incompatible function that are co-expressed at high levels on tumor cells but not on normal tissues, it may be possible to generate reagents that inhibit tumor growth with enhanced selectivity.

The inhibitory effect of endotoxins on growth of human cell lines.

A study was conducted to examine the effect of endotoxin present in periodontal pockets on the proliferation and attachment of human cell lines on the culture plates (Ca9-22 and gingival fibroblasts). The endotoxin was collected from periodontal pockets of anterior teeth in patients with periodontal disease by subgingival irrigation with sterilized distilled water. The solutions obtained were then subjected to hot phenol-water extraction. The collected endotoxin from periodontal pocket and four other kinds of endotoxin obtained commercially as positive controls were added to cell cultures and the numbers of viable cells on the culture plates were counted. Among the commercially available endotoxins used in this study, only 500 micrograms/ml of endotoxin derived from Escherichia coli 0111:B4 significantly decreased the number of attachment cells of Ca9-22 and gingival fibroblasts on the culture plates. Endotoxin from periodontal pockets at 5 micrograms/ml also significantly decreased the numbers of attachment cells of both cell lines.

Clonal growth of bladder cancer cells in a double layer soft agar assay: needles addition of multiple culture supplements

The necessity of multiple culture supplements, characteristic of Hamburger and Salmon (H&S) soft agar assay (1977), for the clonal growth of bladder cancer cells was systematically investigated. One murine and 6 human cell lines were cultivated in different culture supplemented conditions based on the H&S method. All cell lines successfully formed colonies in the absence of supplements. The inclusion of 2-mercaptoethanol to supplement-free medium stimulated the clonal growth of murine (MBT-2) cells, whereas it did not affect the growth of human cell lines. DEAE-dextran suppressed the clonal growth of 5 out of 7 lines. Tryptic soy broth stimulated the growth of 2 human cell lines, whereas it suppressed the other 2 cell lines. The chemosensitivity of MBT-2 cells was not affected by these supplements.

Isolation and analysis of adenovirus type 5 mutants containing deletions in the gene encoding the DNA-binding protein.

A genetic system is described which allows the isolation and propagation of adenovirus mutants containing lesions in early region 2A (E2A), the gene encoding the multifunctional adenovirus DNA-binding protein (DBP). A cloned E2A gene was first mutagenized in vitro and then was introduced into the viral genome by in vivo recombination. The E2A mutants were propagated by growth in human cell lines which express an integrated copy of the DBP gene under the control of a dexamethasone-inducible promoter (D. F. Klessig, D. E. Brough, and V. Cleghon, Mol. Cell. Biol. 4:1354-1362, 1984). The protocol was used to construct five adenovirus mutants, Ad5d1801 through Ad5d1805, which contained deletions in E2A. One of the mutants, Ad5d1802, made no detectable DBP and thus represents the first DBP-negative adenovirus mutant, while the four other mutants made truncated DBP-related polypeptides. All five mutants were completely defective for growth and plaque formation on HeLa cell monolayers. Furthermore, the two mutants which were tested, Ad5d1801 and Ad5d1802, did not replicate their DNA in HeLa cells. The mutant Ad5d1804 encoded a truncated DBP-related protein which contained an entire amino-terminal domain derived from the host range mutant Ad5hr404, a variant of Ad5 which multiplies efficiently in monkey cells. While results of a previous study suggest that the amino-terminal domain of DBP could act independently of the carboxyl-terminal domain to enhance late gene expression in monkey cells, the Ad5d1804 polypeptide failed to relieve the block to late viral protein synthesis in monkey cells. The mutant Ad5d1802 was used to study the role of DBP in the regulation of early adenovirus gene expression in infected HeLa cells. These experiments show that E2A mRNA levels are consistently reduced approximately fivefold in Ad5d1802-infected cells, suggesting either a role for DBP in the expression of its own gene or a cis-acting defect caused by the E2A deletion. DBP does not appear to play a significant role in the regulation of adenovirus early regions 1A, 1B, 3, or 4 mRNA levels in infected HeLa cell monolayers since wild-type Ad5- and Ad5d1802-infected cells showed very little difference in the patterns of expression of these genes.

Growth responses of cell cultures in a galactose medium

Under specific conditions utilized, Eagle's minimum essential medium containing “substantially glucose-free” galactose instead of glucose supported the growth of every cell culture tested with the exception of embryonic cells. Growth of various primary kidney cultures in galactose-EMEM was greater or, at least, equal to that obtained with the same medium containing glucose. Cell lines of non-human origin showed extensive growth in galactose-EMEM and were further stimulated by supplemental pyruvate. Only limited, if any, growth of human cell lines occurred in galactose-EMEM under routine conditions of culture. The growth response of these cells was greatly increased and approached that with glucose if the initial pH of the galactose medium was adjusted in the acid pH range or, with WISH cells, if the galactose medium was supplemented with pyruvate.