Aneuploidy induction in human fibroblasts: comparison with results in Syrian hamster fibroblasts

The susceptibility of human fibroblast cells in culture to neoplastic transformation by chemical carcinogens is appreciably lower than that of rodent fibroblasts. We have proposed that a key step in the neoplastic progression of Syrian hamster embryo fibroblasts is the induction of aneuploidy by carcinogens. It is possible that the different sensitivity to neoplastic transformation of Syrian hamster versus human cells is due to a difference in genetic stability following treatment with chemicals inducing aneuploidy. Therefore, we measured the induction of numerical chromosome changes in normal human fibroblasts and Syrian hamster fibroblasts by 4 specific aneuploidogens. Dose- and time-dependent studies were performed. Nondisjunction, resulting in aneuploid cells with a near-diploid chromosome number, in up to 14-28% of the hamster cells was induced by colcemid (0.1 microgram/ml), vincristine (30 ng/ml), diethylstilbestrol (DES) (1 microgram/ml) or 17 beta-estradiol (10 micrograms/ml). In contrast, human cells displayed far fewer aneuploid (near-diploid) cells, i.e., 8% following treatment with colcemid (0.02 micrograms/ml) or vincristine (10 ng/ml) and only 3% following treatment with DES (6 micrograms/ml) or 17 beta-estradiol (20 micrograms/ml). The doses at which the maximum effect was observed are given. Treatment of human cells induced a higher incidence of cells with a near-tetraploid chromosome number, which was similar to the level observed in treated hamster cells except at the highest doses. These results indicate that human cells respond differently from hamster cells to agents that induce aneuploidy. In particular, nondisjunction yielding aneuploid human fibroblasts with a near-diploid chromosome number was less frequent. The magnitude of the observed species differences varied with different chemicals. The difference in aneuploidy induction may contribute, in part, to species differences in susceptibility of fibroblasts to neoplastic transformation.     

Effect of season and photoperiod on the follicle-stimulating hormone receptors in a subtropical bird

Annual changes in and photoperiodic influence oh the weight of gonads, a parameter of gonadal activity, are much smaller in female birds than in males. Effect of season and photoperiod on the follicle-stimulating hormone receptors in the testis or ovary was studied using a subtropical weaver finch. The number of follicle-stimulating hormone binding sites per unit testicular weight showed a peak in the non-breeding phase; while the total number of binding sites per two testes was maximal in the breeding phase and minimal in the regressive phase. In contrast, seasonal changes in follicle-stimulating hormone binding sites in the ovary were less marked. Exposure to short-day during the breeding phase induced marked decreases in the numbers of binding sites per unit testicular weight and per two testes. These numbers markedly increased after transfer to long-day during the non-breeding phase. However, there was no significant effect of short-day or long-day exposure on follicle-stimulating hormone binding sites in the ovary. These results suggest that photoperiod is an effective environmental factor in the regulation of follicle-stimulating hormone receptors in the testis and the effect is manifested by pronounced changes in the testicular weight during annual breeding cycle.     

Cell cycle dependence of cytotoxicity and clastogenicity induced by treatment of synchronized human diploid fibroblasts with sodium fluoride

To study the cell cycle dependence of cytotoxicity and clastogenicity of sodium fluoride (NaF), synchronized human diploid fibroblasts were treated with NaF during different phases of the cell cycle and analyzed. Exponentially growing cells were synchronized by the following two procedures. (1) The cells were synchronized at G₀/G₁ phase by a period of growth in medium containing 1% serum (low serum medium). (2) The cells were synchronized at the G₁/S boundary by growth in low serum medium, followed by hydroxyurea treatment (Tsutsui et al., 1984a). Synchronized cells were treated with NaF for 3 h during the G₁ phase or G₂ phase, and for each of three 3-h periods during the S phase which lasted 9 h. Cytotoxicity, as determined by a decrease in colony-forming ability, was dependent upon the phase of the cell cycle during which NaF treatment was administered. The highest lethality was induced in when the cultures were treated with NaF during the second or third 3 h of S phase (middle or late S phase, respectively), or G₂ phase. Little lethality was observed in cultures in G₁ phase. Inducibility of chromosome aberrations of the cells following treatment with NaF was also dependent upon the phase of the cell cycle. A significant increase in the incidence of chromosome aberrations was observed only in cultures treated with NaF during early and / or middle S phases of cell cycle. These results suggest that cytotoxicity and clastogenicity of NaF to cultured human diploid fibroblasts are cell cycle dependent, and that the cells in early and middle S phases are more sensitive to the effects.     

Chemical synthesis of insulin-like peptide 1 and its potential role in vitellogenesis of the kuruma prawn Marsupenaeus japonicus

The insulin superfamily comprises a group of peptides with diverse physiological functions and is conserved across the animal kingdom. Insulin-like peptides (ILPs) of crustaceans are classified into four major types: insulin, relaxin, gonadulin, and androgenic gland hormone (AGH)/insulin-like androgenic gland factor (IAG). Of these, the physiological functions of AGH/IAG have been clarified to be the regulation of male sex differentiation, but those of the other types have not been uncovered. In this study, we chemically synthesized Maj-ILP1, an ILP identified in the ovary of the kuruma prawn Marsupenaeus japonicus, using a combination of solid-phase peptide synthesis and regioselective disulfide bond formation reactions. As the circular dichroism spectral pattern of synthetic Maj-ILP1 is typical of other ILPs reported thus far, the synthetic peptide likely possessed the proper conformation. Functional analysis using ex vivo tissue incubation revealed that Maj-ILP1 significantly increased the expression of the yolk protein genes Maj-Vg1 and Maj-Vg2 in the hepatopancreas and Maj-Vg1 in the ovary of adolescent prawns. This is the first report on the synthesis of a crustacean ILP other than IAGs and also shows the positive relationship between the reproductive process and female-dominant ILP.     

Crystal structure of Streptomyces erythraeus trypsin at 2.7 A resolution.

The crystal structure of Streptomyces erythraeus trypsin (abbreviated as SET) has been determined in order to clarify the precise structure of the vicinity of the active site of serine protease and to understand its structure-function relationship. Crystals of SET were prepared at its active pH range (pH 5-10) without any inhibitors which might have affected the circumstances around the active sites. The structure model of SET was made based on the electron density map obtained by the multiple isomorphous replacement method at 3.5 A resolution, and refined by the restrained least-squares method. The current model yields a crystallographic R-factor of 0.272 for 4,968 reflections between 8 and 2.7 A resolution. Though the sequence homology among SET, Streptomyces griseus trypsin and bovine trypsin, 32-37%, is not so high, their overall structures are similar to each other. Comparison of the three molecular structures shows that: 1) the folding of the main chains of the three proteins is essentially the same though there are significant differences on the molecular surface; 2) the spatial arrangements of the catalytic triads in the three proteins are similar to each other; 3) in SET and S. griseus trypsin a short stretch of 3(10)-helix is found through Ala56 to Thr59; His57 in this segment is one important amino acid residue involved in the active sites.     

Mechanism of induction of cellular DNA synthesis by the adenovirus E1A 12S cDNA product

The mechanism of induction of DNA synthesis in quiescent rat 3Y1 cells by the adenovirus E1A gene was investigated using the 3Y1 derivative cell lines g12-21, gn12RB1, and gn12RB2. The g12-21 cells express the E1A 12S cDNA and the latter two cells express both the E1A 12S cDNA and the human retinoblastoma susceptibility (Rb) gene at different levels in response to dexamethasone (dex). The cDNA sequences of E1A-inducible cell cycle-dependent genes, clone 3 and clone 16, were isolated by differential screening of a cDNA library constructed from dex-treated g12-21 cells. The quiescent 3Y1 cells induced c-fos and c-myc expression within 2 h after serum stimulation and expressed clone 16 and clone 3 transiently at around 8 h before the onset of DNA synthesis (10 h). In contrast, the quiescent g12-21 cells treated with dex expressed a high level of E1A at 6 to 8 h after treatment and expressed clone 16 and clone 3 at around 8 h without stimulation of c-fos and c-myc expression, suggesting that E1A bypasses the cell cycle early in G1. The half-maximal rate of DNA synthesis was reached in a much shorter time in dex-treated g12-21 cells (12 h) than in serum-treated 3Y1 cells (18 h), suggesting that E1A also bypasses the cell cycle at the G1/S boundary. The gn12RB1 and gn12RB2 cells were unable to induce DNA synthesis in response to dex presumably due to lower levels of E1A expression, although gn12RB2 but not gn12RB1 cells could express clone 16 and clone 3. These results suggest that the level of E1A required for bypass at the G1/S boundary is higher than that required early in G1.     

A new family of heparin-binding factors: strong conservation of midkine (MK) sequences between the human and the mouse

A retinoic acid responsive gene, MK, specifies for a heparin binding factor termed midkine (MK), which is the initial member of a new protein family involved in regulation of growth and differentiation. A cDNA clone of human MK was isolated from a fetal kidney cDNA library. Human MK mRNA was expressed in PA1 teratocarcinoma cells as well as in the kidney. Sequence analysis of the cDNA clone and of a part of the genomic clone yielded the predicted protein sequence of human MK. Human and mouse MK sequences are highly conserved: 87% of amino acids are identical and all amino acid changes are conservative except for an insertion. Comparison of MK and HB-GAM/pleiotrophin (another member of the family) from various species revealed sequences conserved in the family and those specific for each protein.     

A protein with multiple heme-binding sites from rabbit serum

A 93,000 molecular weight protein (HBP.93) which binds hemin and protoporphyrin IX with high affinity has been isolated from rabbit serum using affinity chromatography on hemin-conjugated agarose. The amino acid composition of this protein is unique in that the proline and histidine contents are remarkably high (16.6 and 9.9 mol %, respectively). A large increase in the absorbance of the Soret region arises from the heme-protein interaction. The spectrophotometric titration showed that the protein can bind 25-35 mol of hemin/mol of protein. The apparent dissociation constant was estimated to be 1-4 X 10(-7) M for hemin at pH 7.4 and approximately 10(-6) M for protoporphyrin IX at pH 9.2. The similarity of the difference spectrum of heme-HBP.93 complex to that of heme-hemopexin complex suggests that a bisimidazol-type coordination of heme iron is involved in the binding. The extremely high capacity of HBP.93 to bind heme is also demonstrated by a large increase in the sedimentation velocity of the protein upon heme binding. The native heme-protein complex migrates faster than the heme-free protein in a polyacrylamide gel at pH 8.8; the increased mobility appears to be due to the charge on the carboxyl groups of the bound heme. Although the use of a hemin-agarose column has failed to reveal a protein of similar size and heme affinity in the sera of a number of other species, including man, the heme-binding properties and high histidine level of the human alpha 2-histidine-rich glycoprotein raise the possibility that the two proteins are related.     

Comparison between mutagenesis in normal and transformed syrian hamster fibroblasts: Difference in the temporal order of HPRT gene replication

A highly tumorigenic subdiploid cell line, BP6T, derived in our laboratory from Syrian hamster embryo (SHE) cells, is amenable to studies of somatic mutation in vitro. Cellular and biochemical characterization of clonally derived BP6T cells resistant to 6-thioguanine (TG^r) or ouabain (Oua^r) demonstrated these mutants to be similar qualitatively to mutants of SHE cells characterized previously (Barrett et al., 1978). BP6T TG^r mutants resistant to 6-thioguanine are cross-resistant to 8-azaguanine, lack HPRT activity, exhibit a low frequency of reversion and arise spontaneously at a rate of 5 × 10⁻⁷ mutants per cell per generation. BP6T Oua^r mutants were shown to be highly resistant to ouabain-mediated inhibition of ⁸⁶Rb influx, indicating an alteration in the Na⁺/K⁺ ATPase. These studies on the BP6T cell line provide the experimental basis for a comparative study of the mutagenic responses of normal, diploid SHE cells versus those of related, but transformed aneuploid cells. Highly synchronized cultures of these 2 cells were mutagenized by pulse treatment with BrdU during different periods of S phase, followed immediately by near-UV irradiation. The induced mutation frequencies so obtained provided information about the temporal order of replication of genes encoding HPRT and Na⁺/K⁺ ATPase in both SHE and BP6T cells. The temporal pattern of replication of Na⁺/K⁺ ATPase gene loci is similar in both cell types, but the temporal order of replication of the HPRT gene is significantly different between SHE and BP6T cells (mid-late S phase, versus early S phase, resp.). This observed difference emphasizes the caution required in the study of mutagenesis and DNA replication using transformed, aneuploid cells under the assumption that the underlying mechanisms are the same for normal, diploid cells.