p53 expression measured by flow cytometry. A comparison of three monoclonal antibodies and the relationship with grade and DNA ploidy in breast cancer

The aim of this study was to quantify p53 expression by flow cytometry. A panel of three monoclonal antibodies: NCL-p53-240, NCL-p53-1801 and NCL-p53-DO7, was tested on breast cell lines and primary breast cancers. The relationships between ploidy, tumour grade and p53 expression for each antibody, were examined. Methodology was assessed using a variety of breast cell lines. Staining patterns were confirmed and the quantification technique qualified. Cytokeratin-positive cells from 58 samples obtained from patients with breast cancer were assayed for DNA content and p53 expression. p53 quantification was performed using calibrated fluorescent beads on cytokeratin-positive cells. Bloom and Richardson grading revealed 20 grade I and 38 grade II/III breast cancers. Examination of fluorescence thresholds showed a positive correlation between grade and DO7 (P=0.003) at a level of 8900 molecules, 240 (P=0.005) at a level of 2900 molecules and 1801 (P=0.005) at a level of 1850 molecules. These levels equated with 34% (DO7), 43% (240) and 43% (1801) of the samples being classified as p53-positive. Examination of ploidy revealed 23 diploid and 35 aneuploid breast cancers. Application of p53 threshold levels on diploid and aneuploid tumours showed correlation between aneuploidy and p53 expression for DO7 at a level of 9000 molecules, 240 at a level of 1900 molecules and 1801 at a level of 1800 molecules. These levels equated with 34% (DO7), 52% (240) and 52% (1801) of the samples being classified as p53-positive. We conclude that measurement of p53 by flow cytometry may be of clinical importance by indicating levels of positivity using fluorescence thresholds. p53 expression has been shown to correlate with both grade and ploidy. Flow-cytometric measurement of p53 may be a useful prognostic assay.This study was supported by the North of England Cancer Research Campaign     

The relationship between flow-cytometric and immunohistochemically detected c-erbB-2 expression,grade and DNA ploidy in breast cancer

Quantification of c-erb B-2 and its relationship with other prognostic markers using flow cytometry has been examined. In this study a level for c-erb B-2 expression above which tumours are classified as positive by flow cytometry has been determined by employment of positive cut-off threshold levels. c-erb B-2 expression by both flow cytometry and immunohistochemistry was studied using the monoclonal antibody NCL-CBII. The relationship of c-erb B-2 quantification by flow cytometry was then compared with ploidy, axillary node status, tumour size and grade. Increased c-erb B-2 expression was seen using flow cytometry. Correlation between immunohistochemistry and flow-cytometry methods just failed to reach significance (P=0.06). Immunohistochemistry revealed a significant relationship between c-erb B-2 expression and aneuploidy (P=0.04). Cytokeratinpositive cells from 110 samples obtained from patients with breast cancer were assayed for DNA content and c-erb B-2 expression by flow cytometry. No correlation was seen between these parameters upon application of Mann Whitney analysis. However, examination of fluorescence thresholds showed a positive correlation between grade and c-erb B-2 expression at a level of more than 3200 molecules (P0.03). At the level of 3600 molecules significance was increased (P=0.004). These levels equated with between 15% and 19% of the samples being classified as c-erb B-2 positive. Application of these cut-off points showed no correlation between c-erb B-2 expression and ploidy, tumour size or axillary node status. Comparison of ploidy and grade showed a significant association (P=0.0015), increased grade correlating with aneuploidy.     

鱼类染色体组操作的研究 Ⅰ.静水压休克诱导三倍体水晶彩鲫

采用静水压休克方法研究了促使第二极体保留诱发三倍体水晶彩鲫的最佳条件。在卵受精后4—5min采用600kg/cm~2或650kg/cm~2的静水压处理3min,不但能导致100％的三倍化,而且胚胎的存活率相当高,孵化率为对照组的90％左右。研究表明,静水压休克是进行鱼类染色体组操作的有效方法,休克的最佳条件易于掌握,处理程序易于标准化。文中讨论了静水压处理的条件与三倍体出现率和胚胎存活率的关系,以及最佳条件下和不适条件下胚胎死亡的原因。     

Multiplicative mononuclear small hepatocytes in adult rat liver: their isolation as a homogeneous population and localization to periportal zone

Adult rat liver contains a minor population of hepatocytes called small hepatocytes (SHs) that are smaller in size and show a higher replicative potential than conventional parenchymal hepatocytes (PHs). However, SHs have been hitherto characterized using a "SH-fraction" that was contaminated with PHs. In the present study, we isolated a PH-free SH-fraction from the adult rat liver using fluorescence-activated cell sorter combined with centrifugal elutriation and characterized the hepatocytes in the fraction. These hepatocytes were designated R3Hs in this study. R3Hs were mononuclear and of lower ploidy. They expressed at high levels genes of Cdc2, connexin 26, hydroxysteroid sulfotransferase, pancreatic secretory trypsin inhibitor, and prostaglandin E2 receptor EP3 subtype. We conclude that SHs dominate the periportal zone in the adult liver, because mRNA or proteins of these genes were exclusively expressed by periportal hepatocytes.     

山茶属的细胞地理学研究

本文收集整理了山茶属的细胞学资料,以细胞地理学的方法探讨该属的起源和演化。在山茶属１４组１１９种植物中有１０组６２种已具染色体数目的报道,５４种已知核型,分别占总数的５２％和４５％。山茶属植物具有稳定的基数（ｘ＝１５）和多变的倍性（２ｘ→８ｘ）。每一个组都有二倍体或全为二倍体;多倍体主要出现在Ｓｅｃｔ．Ｃａｍｅｌｉａ、Ｓｅｃｔ．Ｐａｒａｃａｍｅｌｉａ和Ｓｅｃｔ．Ｔｈｅｏｐｓｉｓ之中,大多分布在山茶属分布区的西部、北部和西北部,呈现出北多南少的趋势。山茶属的核型遵守Ｓｔｅｂｂｉｎｓ的从对称向不对称演化的规律,分布中心靠南的组比靠北的组拥有更对称的核型,表明山茶属从南向北进化和扩散。在已具核型资料的组中,Ｓｅｃｔ．Ａｒｃｈｅｃａｍｅｌｉａ最原始,Ｓｅｃｔ．Ｃａｍｅｌｉａ最进化,包括Ｃ．ｙｕｎｎａｎｅｎｓｉｓ的Ｓｅｃｔ．Ｈｅｔｅｒｏｇｅｎｅａ不可能是山茶属最原始的类群。我们认为山茶属很可能起源于中南半岛,主要向北扩散和进化,在现今的分布中心发生了快速的分化,形成了大量种类     

Polyploidization in embryogenic microspore cultures ofBrassica napus L. cv. Topas enables the generation of doubled haploid clones by somatic embryogenesis

Summary Embryogenic microspore and pollen culture followed by subculture of microspore-derived plantlets enabled the production of clones ofBrassica napus cv. Topas. Flow-cytometric analysis revealed that most microspore- and pollen-derived embryos (pEMs) were haploid initially. Spontaneous diploidization occurred at the globular stage of the pEMs, and was expressed as the relative increase of the 2C and 4C nuclear DNA content. Diploidization occurred throughout various organs of the pEMs and resulted in the formation of haploid and doubled haploid chimerics. In some embryos, nearly all cells were doubled haploid. From early cotyledon stage onward, pure haploid embryos were not observed anymore. At late cotyledon and germination stages, pure doubled haploid embryos and plantlets increased in number. Tetraploid pEMs were found occasionally. A culture regime was established to induce somatic embryos on the pEM-derived young plantlets. The ploidy of the somatic embryos varied highly and tended to be the same as that of the tissue at the initiation site on the pEM-plant. The results show that during the embryogenic development ofB. napus microspores, spontaneous diploidization occurs at globular stage, and increases progressively, resulting in the formation of chimerical haploid and doubled haploid plants as well as pure doubled haploid plants; ploidy neither affects pEM development at embryo developmental stages nor somatic embryogenesis, that starts on young pEM-derived plantlets; doubled haploid somatic embryos can be cloned from single pEM-derived plantlets; and doubled haploid embryos develop to fertile plants.     

Cytogenetics of protoplast cultures of Brachycome dichromosomatica and Crepis capillaris and regeneration of plants

Summary Callus derived protoplasts of Brachycome dichromosomatica (2n=2x=4) and Crepis capillaris (2n=2x=6) have been regenerated into karyologically normal plants, i.e. plants without visible alterations of the diploid chromosome set. However, metaphase analysis of protoplast cultures derived from both callus as well as mesophyll cells showed karyological changes in the overwhelming majority of cells in both species leading to multinucleated, polyploid and aneuploid cells. Furthermore, callus derived protoplasts sometimes exhibited changes at the chromosome level as indicated by translocations. The vast majority of aberrant karyotypes arose from failures during mitosis and cytokinesis, pointing to inadequate microtubules as a possible underlying cause. Karyological events of the kind described herein greatly affect the plating efficiency of isolated protoplasts and the viability of protoplast derived calli. Plant regeneration, although demonstrated in this study for the first time in both species, seems to be limited to rarely occurring, protoplast-derived colonies with a relatively stable genome. Our experiments, performed with chromosomal model species, emphasize the need for controlled, non-mutagenic culture conditions.     

Cytophotometric study and statistical analysis of ploidy evolution in cultured tissues of Nicotiana tabacum

Evolution of the ploidy in four strains of Nicotiana tabacum ev. P 19 was studied by cytophotometry after transformation of the cultured tissues into protoplast suspensions.
The limits of these variations were analysed numerically and statistically. The method used is capable of wide application. It can be used to predict the evolution of the average ploidy level of a strain maintained on a given medium if the average ploidy level of the explant is known. Comparison is made relative to a standard of known ploidy (diploid protoplasts). Increase of the average ploidy level is faster when the explant is mainly diploid (leaf parenchyma) than when it is composed of more heterogeneous tissues as regards ploidy (shoot tip). The appearance of a random drift depends on the composition of the culture medium: it is immediate in one medium (MS₂), but is preceded by a phase of relative stability in another medium (MS₁) which is richer in kinetin.
While the results presented specifically concern the strains cultured on media MS₁ and MS₂, the statistical method employed is applicable to cultures of any species for which one can experimentally justify a linear regression analysis.     

Plant regeneration with maintenance of the endosperm ploidy level by endosperm culture in <Emphasis Type="Italic">Lonicera caerulea</Emphasis> var. <Emphasis Type="Italic">emphyllocalyx</Emphasis>

An endosperm culture of Haskap (Lonicera caerulea var. emphyllocalyx) was established to develop polyploid plants and investigate the regeneration ability of the endosperm. Based on histological analysis of embryo and endosperm development, endosperms at the globular to early torpedo-stages of developing embryos were used to initiate an endosperm culture. Formation of shoot primordia was observed on Murashige and Skoog (MS) medium (Physiol Plant 15:473–497, 1962) containing benzyladenine and indole-3-butyric acid. Shoot primordium formation was confirmed in some genotypes with regeneration frequencies ranging between 1.9 and 10.0%. These proliferated on ½ MS medium containing 2.89 μM gibberellic acid (GA₃), and then elongated and rooted on MS medium containing 0.44 μM BA and 2.89 μM GA₃. These shoots developed into plantlets on ½ MS medium. Plantlets maintained ploidy of the endosperm following flow cytometric analysis, thus confirming that these were derived from the endosperm. These results indicated that endosperms were capable of regeneration.