A novel immunohistochemical method to estimate cell-cycle phase distribution in archival tissue: implications for the prediction of outcome in colorectal cancer

An immunohistochemical method for assessing cell-cycle phase distribution in colorectal resection specimens would enable phase data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in colorectal cancer. In contrast to flow cytometry, an immunohistochemical method would also allow the phase distribution to be examined within morphologically heterogeneous regions of neoplasms. Paraffin sections of normal colon (n = 25), colonic adenoma (n = 15), and colonic adenocarcinoma (n = 30) were analysed by immunohistochemistry using antibodies against markers of cell-cycle entry, Mcm-2 and Ki67, and putative markers of the cell-cycle phase, cyclins D1 and E (putative markers of G1 phase), cyclin A (S phase), cytoplasmic cyclin B1 (G2 phase), and phosphohistone H3 (M phase). The phase specificity of each marker was assessed by examining the degree of co-expression of adjacent phase markers using double-antibody fluorescence confocal microscopy and by comparison with flow cytometric analysis performed on adjacent tissue sections. The S-phase specificity of detectable cyclin A was also assessed in combination with in situ DNA replication using fluorescence confocal microscopy. All cells expressing phase markers co-expressed Mcm-2. Adjacent phase markers were not significantly co-expressed, confirming the relative specificity of these markers in tissue sections of colon. Cell-cycle phase distribution, calculated by immunohistochemistry, compared well with phase analyses obtained by flow cytometry. No cells expressed cyclin A in the absence of active DNA replication. The S-phase labelling index, as defined by detectable cyclin A expression, showed a positive correlation with the Mcm-2 labelling index and increased in the progression from normal colon to adenocarcinoma. In conclusion, a combination of these cell-cycle phase markers can be used to calculate the distribution of cells throughout each phase of the cell cycle in colorectal tissue sections. Detectable cyclin A can be used as a surrogate marker of S phase and may be of value in predicting prognosis and response to adjuvant therapy.     

Role of tumor necrosis factor receptor 1 (p55) in hepatocyte proliferation during acetaminophen-induced toxicity in mice

Hepatocyte proliferation represents an important part of tissue repair. In these studies, TNF receptor 1 (TNFR1) knockout mice were used to analyze the role of TNF-alpha in hepatocyte proliferation during acetaminophen-induced hepatotoxicity. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was associated with proliferation of hepatocytes surrounding the damaged areas, which was evident at 24 h. The cell cycle regulatory proteins, cyclin D1 and cyclin A, were also up regulated in hepatocytes. In contrast, in TNFR1-/- mice, which exhibit exaggerated acetaminophen hepatotoxicity, hepatocyte proliferation, and expression of cyclin D1 and cyclin A, as well as the cyclin dependent kinases, Cdk4 and Cdk2, were reduced. The cyclin-dependent kinase inhibitor p21 was also induced in the liver following acetaminophen administration. This was greater in TNFR1-/- mice compared to WT mice. To investigate mechanisms mediating the reduced hepatic proliferative response of TNFR1-/- mice, we analyzed phosphatidyl inositol-3-kinase (PI-3K) signaling. In both WT and TNFR1-/- mice, acetaminophen caused a rapid increase in total PI-3K within 3 h. Acetaminophen also increased phosphorylated PI-3K, but this was delayed 6-12 h in TNFR1-/- mice. Expression of Akt, a downstream target of PI-3K, was increased in both WT and TNFR1-/- mice in response to acetaminophen. However, the increase was greater in WT mice. Acetaminophen-induced expression of phosphorylated STAT3, a key regulator of cytokine-induced hepatocyte proliferation, was also delayed in TNFR1-/- mice relative to WT. These data suggest that TNF-alpha signaling through TNFR1 is important in regulating hepatocyte proliferation following acetaminophen-induced tissue injury. Delayed cytokine signaling may account for reduced hepatocyte proliferation and contribute to exaggerated acetaminophen-induced hepatotoxicity in TNFR1-/- mice.     

氨基胍通过降低周期素激酶抑制剂P27水平减轻高糖培养系膜细胞肥大程度

目的:研究氨基胍(AG)减轻高糖培养系膜细胞(MC)肥大程度与周期素激酶抑制剂P27的关系.方法:Western 印迹方法测定MC裂解液P27水平,[3H]胸腺嘧啶核苷([3H]TdR)及[3H]亮氨酸([3H]Leu)掺入方法测定MC肥大情况.结果:同正常糖(100 mg/dl)培养相比,高糖(450 mg/dl)培养MC中P27水平增高(P<0.01),MC肥大;[3H]Leu掺入增加及[3H]TdR掺入降低(P<0.01);AG(0.25 mmol/L)降低高糖培养MC中P27水平[(2.5±0.6 ) vs (4.0±0.8),P<0.05],使MC肥大程度减轻;[3H]leu掺入降低,[(3 426±532) vs (4 652±309) cpm/孔,P<0.01],[3H]TdR掺入增加[(1 233±69) vs (543±27) cpm/孔,P<0.01].结论:AG可能通过降低高糖培养MC中P27水平减轻MC肥大程度.     

细胞周期与抗肿瘤治疗展望

各类cyclins、CDKs、CKIs和chk等因素作为细胞周期的调控机制,对细胞的增殖、分化和凋亡具有关键的作用。肿瘤是一类细胞周期性疾病,它们可能会成为抗肿瘤药物的新靶点,达到治疗肿瘤的目的。     

慢性肾炎系膜细胞周期调控蛋白的表达与中医证型关系

为探讨中医证型与慢性肾炎周期控蛋白表达的关系，对３２例慢性肾炎患者进行中医辩证分型，并将肾活检标本分别行常规病理检查及Ｐ２７、ＰＣＮＡ的免疫组化检查。结果：Ⅰ级病理损害以Ｐ２７表达最高，Ⅲ组病理损害以ＰＣＮＡ表达最高，Ｐ２７／ＰＣＮＡ比值与病理分级的等级相关分析ｒ＝－０．６９８４，呈负相关；中医证型与病理分级、Ｐ２７／ＰＣＮＡ比值与病理分级的等级相关分析ｒ分别为０．６９１４、０．６１７４，呈正相关。结论：Ｐ２７、ＰＣＮＡ作为诊断慢性肾炎的系膜细胞增殖或抑制，有其实用性；中医证型与病理分级程度密切相关；进一步推测中医证型的转化，其病理基础与Ｐ２７、ＰＣＮＡ表达相关。     

骨软骨瘤和骨恶性肿瘤cyclinD1和CDK4的表达及意义

目的研究骨软骨瘤和骨恶性肿瘤组织中cyclinD1和CDK4表达及其临床病理意义.方法骨肿瘤组织脱钙后常规石蜡包埋切片ABC免疫组化法.结果15例骨软骨瘤cyclinD1和CDK4表达均阴性,45例骨恶性肿瘤cyclinD1和CK4表达阳性率分别为44%(20/45)和60%(27/45).组织学分级1级和无原发灶外转移骨恶性肿瘤cyclinD1和CDK4阳性率明显低于组织学分级Ⅲ级和原发灶外转移病例,其中分级之间均存在显著差异(P＜0.05);骨恶性肿瘤中cyclinD1和CDK4表达存在密切正相关(P＜0.05).结论cyclinD1和CDK4表达与骨恶性肿瘤生物学行为和预后存在较密切关系,骨恶性肿瘤的分子发病机制可能涉及到CDK4/.cyclinD1调节通路异常,检测骨良性病变cyclinD1和CDK4的表达可能对预防和早期发现骨恶性肿瘤有一定临床意义.     

Cyclin D_1蛋白的表达与鼻咽癌放射治疗的疗效

目的探讨CyclinD1基因蛋白的表达与鼻咽癌放射治疗疗效的相互关系。方法应用抗CyclinD1基因蛋白单克隆抗体 ,采用免疫组化S P法 ,检测 76例鼻咽癌组织标本中CyclinD1基因蛋白的表达情况。将CyclinD1基因蛋白的表达程度分 4个级别 ,应用 χ2 检验放射治疗与基因蛋白表达程度是否相关。结果CyclinD1基因蛋白的表达与鼻咽癌放射治疗疗效明显相关。结论CyclinD1基因蛋白的表达与鼻咽癌放射治疗疗效之间具有明显相关性 ,CyclinD1基因蛋白的表达可作为指导鼻咽癌临床治疗及预后判定的一项参考指标     

Differential regenerative response and expression of growth factors following hepatectomy of variable extent in rats

Abstract: Aims/Background: Liver regeneration is a physiological mechanism which leads to restoration of the hepatic parenchyma following hepatectomy or toxic injury. This process is mediated by a wide variety of cytokines and growth factors. The aim of the present study was to evaluate the influence of hepatectomy extent on the levels of intrahepatic mRNAs for cell-cycle markers and growth factors in rats submitted to a 30%, two-third or 80% hepatectomy. Methods: Cyclins, thymidine kinase and growth factors mRNA levels were quantitatively assessed by RT-PCR at different time points post-hepatectomy (2h, 6h, 12h, days 1, 2, 6). Results: As compared with a two-third hepatectomy, cyclins and thymidine kinase mRNA levels were increased but with a delayed peak at day 2 in the 80% hepatectomy group and showed a progressive increase until day 6 in the 30% hepatectomy group; mRNA levels for HGF or TGFα were increased with a delayed peak at 12 h or day 2 in the 80% hepatectomy group, respectively and this delay was more pronounced in the 30% hepatectomy group with a peak at day 1 or day 6. Conclusion: A regenerative response occurs whatever the extent of hepatectomy but the course of regeneration and expression of growth factors differs according to the volume of resected liver. A better knowledge of these events could improve the clinical results of hepatic resection for primary or metastatic liver disease.     

Cord blood megakaryocytes do not complete maturation, as indicated by impaired establishment of endomitosis and low expression of G1/S cyclins upon thrombopoietin-induced differentiation

Cord blood (CB) has successfully been used as a stem cell source for haemopoietic reconstitution. However, a significant delay in platelet engraftment is consistently found in CB versus adult peripheral blood (PB) or bone marrow transplants. We sought to determine whether or not CB megakaryocytes have reached terminal maturation and, hence, full thrombopoietic potential. A comparative analysis of megakaryocytes cultured from either CB or PB progenitors in the presence of thrombopoietin (TPO) showed a similar differentiation response, although proliferation was 2.4 times higher in CB than in PB cells. Importantly, the TPO-induced ploidy level was notably different: whereas 82.7% of CB megakaryocytes remained diploid (2N) at the end of the culture, more than 50% of PB megakaryocytes had reached a DNA content equal to or higher than 4N. Western blot and flow cytometry analyses revealed that only polyploid PB megakaryocytes expressed cyclins E, A and B, whereas cyclin D3 was detected in both fetal and adult megakaryocytic nuclei. These data suggest that establishment of endomitotic cycles is impaired in CB megakaryocytes, associated with a differential regulation of G1/S cell cycle factors. We believe that the relative immaturity of fetal megakaryocytes could be a contributing factor to the delayed platelet engraftment in cord blood transplantation.