Sexing the human fetus and identification of polyploid nuclei by DNA-DNA in situ hybridisation in interphase nuclei

Samples of human adult lymphocytes, fetal lymphocytes, amniotic fluid cells, and chorionic villus cells were sexed independently by cytogenetics and DNA-DNA in situ hybridisation to a tritiated Y probe. For the in situ hybridisation analysis, the presence of Y bodies (hybridisation bodies) in 100 interphase nuclei were scored after autoradiography. In all, 82/83 samples were sexed in this way (one technical failure) and 78/82 were sexed by both in situ hybridisation and cytogenetics. There was complete agreement between the two methods. There was a considerable variation (40-100%) in the percentage of interphase nuclei with a hybridisation body among the male samples, but very few nuclei from female samples showed significant hybridisation. In situ hybridisation could be used to sex the conceptus when males but not females are at risk for various X-linked genetic disorders and may also be useful for detecting 45,X/46,XY mosaicism or polyploid/diploid mosaicism. This would be particularly useful for direct preparations of chorionic villus samples, which often prove difficult to analyse cytogenetically but offer the best means of avoiding maternal contamination. Some interphase nuclei had more than one hybridisation body, and this was most commonly found among amniotic fluid cells. Comparison of sizes of nuclei with one or two hybridisation bodies strongly suggested that most of the amniotic fluid cell nuclei with two hybridisation bodies were tetraploid.     

Influence ofl-thyroxine,l-triiodothyronine,thyroid stimulating hormone,or estradiol on the cell kinetics of cultured mammary cancer cells

Summary We describe the in vitro influence of 3,5,3′-triiodo-l-thyronine (T₃),l-thyroxine (T₄), a thyroid-stimulating hormone (TSH), and/or estradiol (E₂: chosen as the control of the methodology) on the cell kinetics (cell distribution in the S+G₂+M phases) of mouse MXT and human MCF-7 mammary cancer cells. Experiments were performed by means of a cell image processor, analyzing MCF-7 or MXT cells that had been grown on glass cover slips and whose nuclei had been stained by the Feulgen reaction, which is selective and quantitative (stoichiometric) with respect to DNA. We show that T₃, T₄, and TSH at 0.01 μM dramatically stimulate the cell kinetics of the MXT mouse and the MCF-7 human mammary cancer cell lines. Indeed, the three hormones bring about a significant transient increase in the S+G₂+M fraction as does E₂. Furthermore, our data indicate that E₂ and TSH are antagonistic with regards to MXT or MCF-7 cell kinetics. This work is supported by grants awarded by the IRSIA and the Fonds de la Recherche Scientifique Médicale (FRSM, Belgium).     

Biomechanical interactions of cancer cells with the microvasculature during metastasis

Metastasis is a major, life-threatening complication of cancer. The bloodstream is the most important disseminative route for cancer cells liberated from their parent tumors. Single circulating cancer cells are arrested in the microvasculature, where the vast majority are killed by rapid or slow processes, and the relatively few survivors grow into micrometastases. We review the underlying causes of one type of rapid cancer cell death in the microcirculation, namely, that caused by biomechanical interactions of cancer cells with microvessel walls, which may result in cell surface membrane expansion and lethal rupture. These lethal interactions appear to be important rate-regulators in hematogenous metastasis, and to dictate some aspects of metastatic patterns. Although these are not the only interactions involving cancer cells, in contrast to others involving cellular and humoral defense mechanisms, they have received comparatively little attention.     

第Ⅷ凝血因子基因内限制性片段长度多态性(RFLP)的研究及其在甲型血友病基因连锁分析中的应用

本文系统地研究了广东地区汉族人群中FⅧ:C基因内BclⅠ,XbaⅠ和BgⅡ位点RFLP的基因频率。多态性位点BclⅠ,XbaⅠ及BglⅠ的切点阳性率分别为63.5%、43.5%和100%。对Bcll和Xbal多态性切点连锁情况研究显示,19.5%的Bcll切点阳性纯合子为Xbal切点杂合子,证明联合应用此两位点RFLP可以把甲型血友病基因连锁分析的有效率提高到65.9%。用RFLP连锁分析对两例甲型血友病家系中的女性进行了致病基因携带者检测,对另一例家系进行了基因产前诊断。     

Ras interaction with the GTPase-activating protein (GAP)

Biologically active forms of Ras complexed to GTP can bind to the GTPase-activating protein (GAP), which has been implicated as possible target of Ras in mammalian cells. In order to study the structural features of Ras required for this interaction, we have evaluated a series of mutant ras proteins for the ability to bind GAP and a series of Ras peptides for the ability to interfere with this interaction. Point mutations in the putative effector region of Ras (residues 32-40) that inhibit biological activity also impair Ras binding to GAP. An apparent exception is the Thr to Ser substitution at residue 35; [Ser-35]Ras binds to GAP as effectively as wild-type Ras even though this mutant is biologically weak in both mammalian and S. cerevisiae cells. In vitro, [Ser-35]Ras can also efficiently stimulate the S. cerevisiae target of Ras, adenylyl cyclase, indicating that other factors may influence Ras/protein interactions in vivo. Peptides having Ras residues 17-44 and 17-32 competed with the binding of Ras to E. coli-expressed GAP with IC50 values of 2.4 and 0.9 microM, respectively, whereas Ras peptide 17-26 was without effect up to 400 microM. A related peptide from the yeast GTP-binding protein YPT1 analogous to Ras peptide 17-32 competed with an IC50 value of 19 microM even though the YPT1 protein itself is unable to bind to GAP. These results suggest that determinants within Ras peptide 17-32 may be important for Ras binding to GAP.     

Normal human colon cells suppress malignancy when fused with colon cancer cells

Summary Normal human colon mucosa cells and cells obtained from histologically normal tissues near that cancer were fused with human colon cancer cells. Resultant hybrid populations of normal and malignant cell fusions behaved as nonmalignant cells in culture, were unable to grow in soft agar, did not express tumor-associated antigens, and were nontumorigenic in nude mice. Autofusion of the cancer cell population led to a phenotype intermediate between normal and malignant cells. That is, the cultures had a much lower plating efficiency in soft agar, and the tumors had a longer latency and slower growth rate in nude mice. This is the first cell culture system to demonstrate that normal epithelial cells can suppress malignancy of their autologous cancer cells, and is a prelude to more extensive studies of genetic events involved in malignant conversion of human colonic epithelium. This study was supported by The University of Texas Health Science Center at San Antonio Center for Human Cell Biotechnology and a graduate student stipend (T. J.) from the Department of Cellular and Structural Biology.     

Effects of interleukin-12 on in vitro culture with interleukin-2 of regional lymph node lymphocytes from lung cancer patients

 In the present study, we carried out a functional analysis of regional lymph node lymphocytes (RLNL) from patients with lung cancer after in vitro activation by interleukin-2 (IL-2) and interleukin-12 (IL-12). IL-12 (100 U/ml) enhanced both the proliferation and cytotoxic activity of RLNL in a culture with low doses of IL-2 (5 – 10 JRU/ml). After comparing an RLNL culture with a low dose of IL-2 alone, a higher proportion of CD8⁺ cells and CD56⁺ cells and a lower proportion of CD4⁺ cells were found in the culture with both IL-12 and a low dose of IL-2. Such a combination of the cytokines effectively activated RLNL in terms of the expression of IL-2 receptors. In the culture condition of IL-12 and a low dose of IL-2, a synergistic effect was observed in the production of such cytokines as interferon γ, tumor necrosis factor α (TNFα), and TNFβ, as well as in tumor cytotoxicity. However, the addition of IL-12 inhibited the cytotoxicity of RLNL in the culture with a high dose of IL-2 (100 JRU/ml). This inhibition is considered to be partially due to the endogenous production of TNFα by lymphocytes, because the neutralization of TNFα bioactivity partially restored the cytotoxic activities of RLNL. Furthermore, in the presence of hydrocortisone, IL-12 synergistically enhanced the cytotoxic activity of RLNL cultured with a high dose of IL-2. These results provide useful information about the improvement of adoptive immunotherapy against cancer using RLNL. Received: 2 February 1996 / Accepted: 30 July 1996