Deletion analysis of protein kinase c inactivation by calphostin C

Protein kinase C (PKC) undergoes specific inactivation by nanomolar concentrations of calphostin C. Both PKC-α (a Ca²⁺-dependent conventional isoform) and PKC-α (a Ca²⁺-independent novel isoform) are similarly inactivated by calphostin C (75–100 nM produced 50% inhibition), suggesting that inactivation requires a site common to both classes of PKC. We therefore performed studies to identify a critical region in the regulatory domain of PKC-α required for inactivation by calphostin C. A series of N-terminal–truncation mutants of bovine PKC-α expressed in Saccharomyces cerevisiae was tested with 500 nM calphostin C, a concentration sufficient to inactivate wild-type PKC-α by 80–90%. This concentration was as effective with mutant proteins containing deletions of up to 91 amino acid (aa) residues from the amino terminus (ND91), whereas a mutant protein truncated by 140 aa (ND 140) was inactivated by only 20%. These findings imply that the aa sequence 92–140 is a structural determinant of PKC-α inactivation by calphostin C. This sequence contains one of the phorbol ester-binding sites (aa 102–144), which is highly conserved among most PKC isoforms including PKC-α. In addition to aa 92–140, PKC-stimulating cofactors (phosphatidylserine, phorbol ester, and Ca²⁺) are required for inactivation by calphostin C even in the case of PKC mutants that do not require these cofactors for enzymatic activity. These results suggest that cofactors provide a template that is required for productive interaction of PKC and the inhibitor. The significance of the proposed proximity effect of calphostin C action is discussed. © 1995 Wiley-Liss Inc.     

鼻咽癌ＫＤＲ受体信号传导相关蛋白的表达及意义*

目的　分析鼻咽癌组织中ＫＤＲ及其下游信号分子ＰＫＣ-β和ｐ-ＥＲＫ的表达,并探讨上述蛋白表达与鼻咽癌患者临床特征和生存期的关系。方法　免疫组织化学法检测１１０例鼻咽癌和３０例鼻咽良性病变石蜡切片中ＫＤＲ、ＰＫＣ-β和ｐ-ＥＲＫ的表达情况,用Ｓｐｅａｒｍａｎ法分析它们之间的相关性,Ｋａｐｌａｎ-Ｍｅｉｅｒ法计算生存情况,行Ｌｏｇ-ｒａｎｋ检验和Ｃｏｘ比例风险模型分析。结果　鼻咽癌组织中ＫＤＲ、ＰＫＣ-β和ｐ-ＥＲＫ阳性表达率分别为８８.２％（９７／１１０）、６３.６％（７０／１１０）和５１.８％（５７／１１０）,而在鼻咽良性病变阳性表达率低。鼻咽癌组织中ＫＤＲ表达与ＰＫＣ-β及ｐ-ＥＲＫ表达呈正相关（ｒ＝０.２９７,Ｐ＝０.００２;ｒ＝０.３３６,Ｐ＜０.００１）,ＰＫＣ β表达与ｐ ＥＲＫ表达呈正相关（ｒ＝０.３０１,Ｐ＝０.００１）。ＫＤＲ和ｐ-ＥＲＫ表达与原发肿瘤的侵犯范围相关（Ｐ＜００５）;ＰＫＣ β和ｐ ＥＲＫ表达与临床分期相关（Ｐ＜０.０５）;ＫＤＲ和ＰＫＣ-β表达与肿瘤局部复发或远处转移相关（Ｐ＜０.０５）。３种蛋白表达均与总生存不佳相关（Ｐ＜０.０５）。Ｃｏｘ多因素相关分析显示年龄＞５０岁为不良预后因素（Ｐ＜０.０５）。结论　ＫＤＲ及其下游信号分子ＰＫＣ-β和ｐ-ＥＲＫ在鼻咽癌中表达,与鼻咽癌患者的临床特征和预后关系密切。     

Effect of serum starvation on expression and phosphorylation of PKC-α and p53 in V79 cells: Implications for cell death

The effect of serum starvation on the expression and phosphorylation of PKC-α and p53 in Chinese hamster V79 cells was investigated. Serum starvation led to growth arrest, rounding up of cells and the appearance of new PKC-α and p53 bands on Western blots. Prolonged incubation (≥48 hr) in serum-deprived medium led to cell detachment and death. Moving cells to fresh medium containing 10% serum before, but not after, cell detachment reversed the changes observed in PKC-α and p53, and also prevented later cell detachment. Radiolabelling studies showed that the higher-molecular-weight PKC-α and p53 bands result from increased phosphorylation, while a lower-molecular-weight PKC-α band reflects newly synthesized protein. Immunocomplex kinase assays have shown that the increased phosphorylation of PKC-α is associated with its increased activity. To study the relationship between PKC-α, p53 and cell death, cells were treated either with TPA, to down-regulate PKC or with staurosporine, to inhibit PKC activity. Staurosporine, a potent PKC inhibitor and inducer of programmed cell death, caused the appearance of new PKC-α and p53 bands similar to those induced by serum starvation. If serum starvation was preceded by prolonged (48 hr) TPA treatment to down-regulate PKC-α, cell detachment and death did not take place within the same time frame. Intracellular fractionation of cells demonstrated that increased expression of PKC-α and the appearance of the associated higher and lower molecular-weight bands occurred in the nucleus. These data highlight the association of PKC-α and p53 with cellular events leading to cell death. Int. J. Cancer 80:400–405, 1999. © 1999 Wiley-Liss, Inc.     

Proliferation of diploid hepatocytes and nonparenchymal (oval) cells during rat-liver regeneration in the presence of 2-acetylaminofluorene

Treatment of rats with the carcinogen 2-acetylaminofluorene (2-AAF) during liver regeneration (Solt-Farber protocol) induced a selective outgrowth of diploid, gamma-glutamyltranspeptidase (GGT)-positive hepatocytes (3-4 times increase) as well as of nonparenchymal (oval) liver cells. After cessation of treatment the oval cells rapidly disappeared, while the population of diploid, GGT-positive hepatocytes declined more slowly over the subsequent ten weeks. In animals pretreated with the initiating carcinogen diethylnitrosamine (DEN) a large fraction of the diploid, GGT-positive hepatocytes persisted. The results differ from those obtained with our standard, sequential treatment protocol (2-AAF given after completed regeneration), where there is no hyperproliferation of oval cells and where GGT-positive hepatocytes are found only in DEN-pretreated animals (Saeter et al, Carcinogenesis 9: 581-587, 1988). Different experimental models of liver carcinogenesis may thus present different patterns of liver cell proliferation, which should be taken into account when general hypotheses on the cellular origin of liver cancer are proposed.     

Increased susceptibility to oxidative DNA damage in regenerating liver

Spontaneous and hepatocarcinogen (2-nitropropane, 2-NP)-inducedlevels of 8-hydroxy-2'-deoxyguanosine (oh⁸dG) in the nuclearDNA of regenerating liver [24, 48, 72 h and 7 days after partialhepatectomy (PH)] of male Sprague-Dawley rats were analysedfor the verification of a hypothesis that the high susceptibilityof proliferating hepatocytes to DNA damage is related to thewell-known high susceptibility to carcinogens after PH. Interestingly,the spontaneous level of nuclear oh⁸dG in regenerating liverwas significantly decreased (P < 0.01) at 48 h after PH (1.05± 0.31 oh⁸dG/10⁵dG) compared with the level in normalrats (1.90 ± 0.41). 2-NP induced an oh⁸dG level of 4.49± 0.86 in nuclear DNA of rat liver without PH. However,in rats administered 2-NP (injections were performed 6 h beforeeach sacrifice) after PH, the oh⁸dG level was significantlyhigher at 24 h (5.45 ± 1.41, P < 0.05), 48 h (5.85± 0.88, P < 0.01) and 72 h (5.67 ± 1.07, P< 0.05) after PH than those with 2-NP exposure alone. Therefore,it is suggested that nuclear DNA in proliferating hepatocytesis in a stage susceptible to exogenous attack by 2-NP, and consequentlythis phenomenon might be related to the induction of hepatocarcinogenesisafter PH.     

氮杂胞苷对射线诱导大鼠Ⅱ型肺上皮细胞系上皮间质转换的影响

目的 探讨氮杂胞苷对射线诱导大鼠Ⅱ型肺上皮细胞系(RLE-6TN)上皮间质转换的影响,并探究其发生机制,为放射性肺纤维化提供潜在的药物治疗实验依据。方法 体外培养RLE-6TN细胞根据实验目的分为对照组(C)、照射组(R)、氮杂胞苷组(A)、照射加氮杂胞苷组(R+A)。细胞超微结构改变采用透视电子显微镜鉴定,细胞形态学改变采用倒置相差显微镜观察。运用实时荧光定量RT-PCR检测E-cadherin和α-SMA在mRNA水平的表达,运用蛋白印迹法检测E-cadherin、GSK3β、p-GSK3β(ser9)在蛋白水平的表达。单因素方差分析组间差异。结果 R组细胞形态趋于纺锤形,而R+A 变化不明显。R组嗜锇性板层小体最终消失。RT-PCR提示在mRNA水平R组相对C组E-cadherin表达减少[(0.23±0.06)∶(1.00±0.00),P=0.002]、α-SMA表达增多[(2.91±0.01)∶(1.00±0.00),P=0.000],而R+A组相对R组E-cadherin表达增加[(0.47±0.05)∶(1.00±0.00),P=0.024]、α-SMA表达减少[(2.50±0.02)∶(1.00±0.00),P=0.037)]。蛋白印迹法结果显示R组相对C组E-cadherin在蛋白水平表达下调[(0.07±0.01):(0.48±0.02),P=0.028]、p-GSK3β表达上调[(0.85±0.04)∶(0.23±0.03),P=0.031],而R+A组相对R组E-cadherin表达下降[(0.25±0.00)∶(0.07±0.01),P=0.024)]、p-GSK3β表达上调程度明显减少[(0.39±0.03)∶(0.85±0.04),P=0.014]。结论 X射线可引起上皮细胞上皮间质转换,而氮杂胞苷通过抑制p-GSK3β活性来抑制放射诱导的RLE-6TN细胞上皮间质转换的发展。     

组蛋白去乙酰化酶抑制剂LBH589逆转多发性骨髓瘤细胞耐药机制的体外研究

 【摘要】　目的　探讨新一代组蛋白去乙酰化酶抑制剂LBH589作用于多发性骨髓瘤（MM）耐药细胞产生的相关基因表达变化,分析其逆转MM细胞耐药的机制。方法　采用Western blot法分析LBH589单药（0、20、50 nmol／L）及50 nmol／L LBH589联合5 μmol／L地塞米松作用MM1R细胞24 h后组蛋白 H4乙酰化的表达程度及bcl-X基因的表达变化。采用实时荧光定量PCR分析LBH589（0、20、50 nmol／L）及50 nmol／L LBH589联合5 μmol／L地塞米松分别作用MM1R细胞24、48 h后TOSO基因的表达变化。结果　Western blot分析显示不同浓度LBH589单药及联合地塞米松作用MM1R细胞24 h后随着药物浓度的升高,组蛋白 H4乙酰化的程度逐渐上调[（0.205±0.008）%、（0.346±0.009）%,（0.580±0.053）%、（0.986±0.012）%,F＝992.957,P＝0.032];bcl-X基因的表达则逐渐下调[（1.210±0.160）%、（0.930±0.036）%、（0.730±0.017）%、（0.488±0.029）%,F＝56.432,P＝0.028],变化呈剂量依赖性,且联合组的作用效果强于单药组（均P＜0.05）。实时荧光定量PCR分析显示不同浓度LBH589单药及联合地塞米松作用MM1R细胞24、48 h后随着药物浓度的增加和时间的延长,TOSO基因的表达逐渐下调[24 h：（1.00±0.00）%、（0.55±0.01）%、（0.47±0.01）%、（0.38±0.01）%,F＝1006.344,P＝0.040;48 h：（1.00±0.00）%、（0.39±0.04）%、（0.05±0.01）%、（0.03±0.00）%,F＝2383.977,P＝0.045],变化呈剂量-时间依赖性,且联合组的作用效果强于单药组（均P＜0.05）。结论　组蛋白去乙酰化酶抑制剂LBH589通过影响bcl-X及TOSO基因的表达,恢复MM1R对地塞米松的敏感性,促进组蛋白乙酰化,诱导细胞凋亡,达到治疗肿瘤的目的。     

IL-1基因多态性对胃粘膜TNF-α、NF-κB的表达和活性的影响

 目的 探讨IL-1的基因多态性影响H.pylori感染后胃癌发生的机制。 方法 通过多态性分析和血清H.pylori抗体测定,收集105例癌低发区的学生作为研究对象,其中IL-1-511T/T 基因型32名（13名H.pylori+）,C/C基因型36名（15名H.pylori+）,C/T基因型37名（14名H.pylori+）。 结果 在胃体组织,H.pylori阴性个体TNF-α和NF-κB表达量极少,而且IL- 1B-511各基因型间也无显著差异。在H.pylori阳性个体,胃体部TNF-α和NF-κB mRNA有较为明显的表达,IL-1B-511T/T基因型个体比C/T和C/C基因型明显增加,分别为1.20±0.17 vs. 0.87±0.18和0.94±0.16;1.10±0.16 vs 0.90±0.15和0.97±0.17,F=33.4和12.9,P=0.0001和0.001。H.pylori阴性的NF-κB mRNA活性很低。在H.pylori阳性个体,NF-κB 活性显著增加。而且IL-1B-511T/T基因型个体比C/T和C/C基因型明显增加,分别为0.99±0.12vs 0.89±0.15和0.90±0.14,F=5.6,P=0.005。 结论 H.pylori感染促进TNF-α和NF-κ B mRNA表达,上调NF-κB活性,在IL-1B-511T/T基因型个体更明显。提示H.pylori感染后,存在IL-1B基因多态性→IL-1β↑→炎症细胞因子↑→胃癌发生的完整通路。     

Appropriate Methodology for EBRT and HDR Intracavitary/Interstitial Brachytherapy Dose Composite and Clinical Plan Evaluation for Patients With Cervical Cancer

PurposeThis study assessed the appropriateness of full parameter addition (FPA) methods with respect to the 3-dimensional deformable dose composite method for evaluating combined external beam radiation therapy (EBRT) and intracavitary brachytherapy (ICBT).Methods and MaterialsA total of 22 patients who received EBRT and high-dose-rate ICBT were retrospectively evaluated. Split-ring and tandem applicators were used for all patients. Additional interstitial needles were used for 5 patients to supplement the implant. Deformable image registrations were performed to deform the secondary EBRT and ICBT planning computed tomography (CT) images onto the reference CT from the third fraction of ICBT. The Dice similarity coefficient was used to evaluate the quality of deformable registration. Doses were transferred to the reference CT, scaled to the equivalent dose in 2-Gy fractions and combined to create the dose composite. Eight dose-accumulation methods were evaluated and compared. D_2cc and D_0.1cc for organs at risk were investigated.ResultsThe differences in D_2cc for rectum, bladder, sigmoid, and bowel between the FPA method for whole-pelvis EBRT and ICBT, calculated using an old American Brachytherapy Society worksheet (FPA_E_h + I_old) and deformable composite for EBRT with boosts and ICBT (Def_E + B + I) were –2.19 ± 1.37 Gy_{α/β = 3}, –0.64 ± 1.13 Gy_{α/β = 3}, –2.06 ± 2.71 Gy_{α/β = 3}, and –1.59 ± 0.89 Gy_{α/β = 3}, respectively. The differences in D_2cc for rectum, bladder, sigmoid, and bowel between the new ABS worksheet (FPA_E_h + B + I_abs) and the Def_E + B + I method were 1.21 ± 1.22 Gy _{α/β = 3}, 1.93 ± 1.38 Gy_{α/β = 3}, 0.72 ± 1.12 Gy_{α/β = 3}, and 1.19 ± 1.46 Gy_{α/β = 3}, respectively. Differences in dose-volume histogram parameter values among Def_E + B + I and other FPA methods were not statistically significant (P > .05).ConclusionsCompared with the FPA-based method, deformable registration–based dose composites demonstrated lower OAR D_2cc and D_0.1cc values; however, the differences were not statistically significant. The current ABS-recommended FPA-based sheet can serve as an acceptable plan evaluation tool for clinical purposes.     

Antitumor Effect of Recombinant Human Interleukin-1β Alone and in Combination with Natural Human Tumor Necrosis Factor-α

In order to investigate the antitumor effect of recombinant human interleukin-1β (IL-1β) alone and in combination with natural human tumor necrosis factor-α (nHuTNF-α), we used female BDF₁ mice bearing Lewis lung carcinoma (3LL). IL-1β showed an antiproliferative effect against pulmonary metastatic tumors of 3LL in a dose-dependent manner. We observed 19.6 ± 6.6, 18.6 ± 5.3, 14.1 ± 4.4 and 13.0 ± 6.0 metastatic tumors at doses of 0.5, 1.0, 2.5 and 5.0 μg IL-1β/mouse/day by daily intravenous administration (the number of metastatic tumors of the control group was 26.3 ± 8.2). Similar results were obtained by intraperitoneal administration, but in this case, mice showed a marked decrease of body weight. When IL-1β was administered in combination with nHuTNF-α, pulmonary metastatic tumors decreased much more than when IL-1β was administered alone. When the control group had 18.6 ± 12.7 metastatic tumors, the nHuTNF-α group had 12.3 ± 3.9 and the IL-1β group had 12.8 ± 8.0, the group which was administered both cytokines had a significantly decreased number of 5.6 ± 3.3 metastatic tumors. This antiproliferative effect of IL-1β in combination with nHuTNF-α was reduced by the intravenous administration of anti-asialo GM₁ antibody and carrageenan. The number of metastatic tumors was increased from 8.9 ± 8.0 to 18.8 ± 11.4 by anti-asialo GM₁ antibody and from 9.5 ± 6.8 to 28.0 ± 12.3 by carrageenan. It was suggested that asialo GM₁-positive cells and macrophage were two of the most important effectors of the antiproliferative effect of IL-1β and TNF-α.     

Effect of vitamin E on the induction and evolution of enzyme-altered foci in the liver of rats treated with diethylnitrosamine

The effects of dietary vitamin E (VE) on the steps of hepatocarcinogenesis,the induction and growth of -glutamyltranspeptidase (GGT)-positivefoci and their evolution into persistent nodules, were analyzedin the liver of rats treated with diethylnitrosamine (DEN).The induction of GGT-positive foci was inhibited by a diet containing0.36–1.5% VE given after initiation with 200 mg/kg bodyweight (b.w.) DEN for 6 weeks with partial hepatectomy (PH)on week 3. The numbers and areas of GGT-positive foci were enhancedby diets containing 036 and 0.72% VE, given for 1 week afterinitiation with 10 mg/kg b.w. DEN and PH, followed by selectionby 0.02/ 2-acetylaminofluorene (AAF) and carbon tetrachloride(CCl⁴), but these were not enhanced by a diet containing 1.5%VE. Remodeling of hyperplastic nodules was not affected by thediet containing 0.72% VE given after initiation with DEN andselection for 12 weeks. The staining characteristics of GGTwere different between remodeling and persistent nodules, exceptfor those of the glutathione-S-transferase placental form (GST-P).The results obtained suggest that VE could prevent the veryearly events during hepatocarcinogenesis, the induction of phenotypicallyaltered foci, but could no longer affect the later stages, theevolution of foci into persistent nodules.     

Genetic Polymorphism in TNF-α-308 G/A and TNF-β +252 A/G,as Prognostic Biomarker in Breast Cancer Patients among Indian Population

Background: Cytokines are the key regulator molecules that modulate immune response. Tumor necrosis factor (TNF- α-308 G/A and TNF-β +252 A/G ) are inflammatory cytokine that control the progression of several types of cancer. They play a vital role in both tumor progression and destruction based on their concentrations. The role of TNF-α-308 G/A and TNF-β +252 A/G gene polymorphism in the etiology of breast cancer (BC) is not clearly understood. Therefore, present study investigates the association of TNF-α -308 G/A and TNF-β +252 A/G and the clinical features with Breast cancer patients. Methods: In a case- control study, we have investigated 150 breast cancer patients and 300 age and ethnically matched healthy controls for duration of 3 years from North India. Promoter polymorphisms of tumor necrosis factor gene (TNF-α -308 G/A and TNF-β +252 A/G) were genotyped using allele specific oligonucleotide polymerase chain reaction ASO and restriction fragment length polymorphism (PCR-RFLP). The associations were evaluated by calculating the pooled odds ratio (OR) with 95% confidence interval (95% CI) using SPSS. Results: Patients with different clinico-pathological variables and healthy controls were analyzed. Significant association was observed in A allele of TNF-α -308 G/A in breast cancer patients as compared to healthy controls (p<0.0001). However, no association was seen in TNF-β +252 A/G both at genotypic and allelic level. The GG genotype of TNF-β +252A/G is higher in grades III (p<0.01) patients. Conclusion: Our results suggest that TNF-α-308G/A polymorphism showed significant association with breast cancer patients.     

肺癌组织中PKC-α蛋白的表达与肺癌临床病理特征的关系

  目的 检测肺癌组织中PKC-α（protein kinase C-alpha）蛋白的表达，探讨PKC-α蛋白与肺癌的组织类型、分化程度肺癌临床病期和淋巴结转移的关系。方法 应用免疫组化链霉茵抗生物素蛋白-过氧化物酶法（SP法）和荧光染色、激光扫描共聚焦技术，检测60例肺癌组织（肺癌组）、17例肺良性病变的正常肺组织（对照组）中PKC-α蛋白表达。结果 肺癌组和对照组PKC-α蛋白表达的阳性率分别为76．7％（46／60）和17．6％（3／17），两组间差异具有高度显著性（P〈0．01）。PKC-α蛋白表达在肺癌不同病理类型即鳞癌和腺癌中，差异无显著性（P〉0．05）。在高分化、中分化和低分化肺癌之间比较，PKC-α蛋白表达阳性率差异亦无显著性（P〉0．05）。PKC-α蛋白在ⅠA＋ⅠB、ⅡA＋ⅡB和ⅢA＋ⅢB期阳性率分别为50．0％、82．6％和90．5％，阳性率随TNM分期而上升，差异有显著性（P〈0．05）。PKC-α阳性率在N0和N1～3肺癌分别为42．1％、92．7％，两者差异有高度显著性（P〈0．01）。41例伴有淋巴结转移的病例中，PKC-α蛋白表达在肺原发灶和淋巴结转移灶之间比较，差异无显著性（P〉0．05）。结论 肺癌组织中存在PKC-α蛋白的高表达；PKC-α蛋白的高表达与肺癌组织类型和分化程度无关，而与肺癌病期、淋巴结转移有密切关系。     

Correlation between clastogenicity and promotion activity in liver carcinogenesis by N-hydroxy-2-acetylaminofluorene, N-hydroxy-4'-fluoro-4-acetylaminobiphenyl and N-hydroxy-4-acetylaminobiphenyl

N-Hydroxy-2-acetylaminofluorene (N-OH-AAF), N-hydroxy-4'-fluoro-4-acetylaminobiphenyl (N-OH-FAABP) and N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) were compared for their initiation and promotion activity in the rat liver using a modified Solt-Farber system. N-OH-AAF, N-OH-FAABP and N-OH-AABP showed comparable initiation capacity when administered to male Wistar rats at a dose of 30, 120 and 120 mumol/kg respectively, 24 h after a two-thirds partial hepatectomy (PH). In contrast, only N-OH-AAF was very effective as promoter when administered to rats previously initiated with diethylnitrosamine. This was evidenced by a high number of large gamma-glutamyltranspeptidase-positive (GGT+) foci occupying a high percentage (22%) of liver volume. N-OH-FAABP was a much weaker promoter, resulting in smaller foci and lower percentage (4%) of GGT+ liver volume. The incomplete carcinogen N-OH-AABP was totally ineffective as promoter in our model. A similar difference was seen in the clastogenicity of these carcinogens in rat liver in vivo as measured by the formation of micronuclei: N-OH-AAF was far more clastogenic than N-OH-FAABP, which in turn was more clastogenic than N-OH-AABP. We have recently shown that N-acetylated deoxyguanosine adducts are responsible for clastogenicity of N-OH-AAF and may be important for promotion. DNA adduct analysis after injection of 120 mumol/kg of tritium-labeled N-OH-FAABP or N-OH-AABP, 24 h after PH, showed that N-acetylated adducts to C8 of deoxyguanosine are also formed from these structurally related liver carcinogens. However, the formation of these adducts from N-OH-FAABP and N-OH-AABP was approximately 8 and approximately 5% of the formation of dG-C8-AAF after injection of 25 mumol/kg N-OH-AAF. These data show that for the structurally related liver carcinogens N-OH-AAF, N-OH-FAABP and N-OH-AABP, clastogenicity does not predict initiating efficacy but correlates with promotion activity. Possibly, N-acetylated adducts to C8 of deoxyguanosine are involved in both clastogenicity and promotion.     

Expression of retinoic acid α and β receptor genes in liver and hepatocellular carcinoma

cDNA probes for human retinoic acid receptors α and β (RARα and RARβ) were modified for use as specific hybridization probes to study hepatocellular carcinomas (HCC) and cell lines, liver regeneration, and fetal development. RARβ mRNA was detected at low levels in adult liver and rose markedly during the early phase of liver regeneration. RARβ mRNA was present at very low levels in HCC and was not detected in fetal liver. In contrast, RARα mRNA was present at low levels in normal liver, but showed a marked elevation in several HCCs and cell lines. Growth of cell lines was altered by retinoic acid (RA), but the effects could not be predicted by the levels of either RARα or RARβ mRNA. However, the response correlated with cell phenotype. Three cell lines with an adult phenotype (high albumin and low α-fetoprotein) were inhibited by RA, two undifferentiated lines showed moderate growth stimulation, and two of three cell lines that had high levels of α-fetoprotein were markedly stimulated by RA.     

Pro-transforming growth factor-alpha processing in human colon carcinoma cells: Role of protein kinase C

The human colon cancer cell lines HCT 116 (poorly differentiated) and CEO (well differentiated) express the mitogenic peptide transforming growth factor alpha (TGF-α). The secretion of TGF-α was enhanced by phorbol 12-myristate 13-acetate (PMA), indicating the possible role of protein kinase C (PKC) in the formation of mature TGF-α. Cells were metabolically labeled with ^3SS-cysteine and the formation of the mature 6 kDa TGF-α polypeptide from the 17 kDa pro-TGF-α precursor was determined. The conversion of pro-TGF-α was complete in 2–4 hr with the HCT 116 cells showing faster kinetics of TGF-α formation than GEO cells. HCT 116 cells secreted more TGF-α than GEO cells and the rate and extent of formation of TGF-α was enhanced by PMA in both cell lines. The expression of several PKC isozymes by HCT 116 and GEO cells was examined by immunoblotting. The expression of all isozymes examined was higher in HCT 116 cells compared with GEO cells. Calphostin C, an inhibitor of PKC, reduced the enzyme activity and significantly inhibited the PMA-induced secretion of TGF-α by both cell lines. Two agonists of PKC that act on specific PKC isozymes, thymeleatoxin and 12-deoxyphorbol 13-phenylac-etate 20-acetate (dPPA), stimulated the release of TGF-α into the medium to the same extent as PMA. Since dPPA has been reported to stimulate PKC-4bT1 specifically, our results suggest a potential role for PKC-β in the processing of pro-TGF-α by these 2 human colon carcinoma cell lines. © 1995 Wiley-Liss, Inc.     

Efficacy of neoadjuvant therapy with trastuzumab concurrent with anthracycline- and nonanthracycline-based regimens for HER2-positive breast cancer

BACKGROUND:

The aim of this study was to evaluate the pathologic complete response (pCR) rates and relapse‐free survival (RFS) and overall survival (OS) of patients receiving neoadjuvant systemic therapy (NST) with trastuzumab in combination with an anthracycline‐ or nonanthracycline‐based regimen.

METHODS:

In this retrospective nonrandomized study, the authors reviewed records of 300 patients with HER2‐positive breast cancer treated with either sequential paclitaxel and trastuzumab and FEC75 in combination with trastuzumab (PH‐FECH) or docetaxel, carboplatin, and trastuzumab (TCH). The Kaplan‐Meier product‐limit method was used to estimate RFS and OS rates. Logistic regression models and Cox proportional hazards models were fit to determine the associations between NST, pCR, and survival.

RESULTS:

There was no significant difference in the decline in cardiac ejection fraction; however, patients who received PH‐FECH had fewer cardiac comorbidities at baseline (P = .002). pCR rates were 60.6% and 43.3% for patients who received PH‐FECH (n = 235) and TCH (n = 65), respectively (P = .016). Patients who received PH‐FECH were 1.45 times more likely to have a pCR (odds ratio [OR], 1.45; 95% confidence interval [CI], 1.06‐1.98; P = .02). Three‐year RFS rates were 93% and 71% (P < .001), and 3‐year OS rates were 96% and 86% (P = .008) for patients who received PH‐FECH and TCH, respectively. Patients who received PH‐FECH had a lower risk of recurrence (hazard ratio [HR], 0.27; 95% CI, 0.12‐0.60; P = .001) and death (HR, 0.37; 95% CI, 0.12‐1.13; P = .08) than those treated with TCH.

CONCLUSIONS:

The type of NST in HER2‐positive breast cancer is predictive of pCR rate independent of disease and patient characteristics. Although TCH is active, PH‐FECH shows a higher pCR rate and RFS advantage. Cancer 2012. © 2011 American Cancer Society.     

Steroid receptor expression in thymomas and thymic carcinomas

BACKGROUND:

Although protein expressions of glucocorticoid receptor (GR), estrogen receptors (ERα and ERβ), progesterone receptor A (PgR‐A), and androgen receptor (AR) were shown to play roles in the growth and differentiation of normal thymus and thymic tumors, to the authors' knowledge their association with patient characteristics and prognosis has yet to be determined.

METHODS:

A series of 140 thymic epithelial tumors (57 type A + AB thymomas, 40 type B1 + B2 thymomas, 6 type B3 thymomas, and 37 thymic carcinomas) were examined for GR, ERα, ERβ, PgR‐A, and AR expression using immunohistochemistry. In addition, the correlation between expression of these hormone receptors and clinicopathologic factors and overall survival (OS) was assessed.

RESULTS:

GR and ERβ demonstrated a high rate of expression in thymomas and thymic carcinomas (82.9% and 76.4%, respectively), whereas rates of ERα, PgR‐A, and AR expression were low (13.6%, 0.71%, and 23.6%, respectively). A significant correlation (P < .05) was found between ERα expression and tumor size and between ERβ expression and tumor stage. Multivariate analyses revealed that histologic subtype (P = .0039), tumor stage (P = .0012), and GR expression (P = .0025) were significantly correlated with the 10‐year OS rate.

CONCLUSIONS:

GR and ERβ demonstrated high rates of expression in thymomas and thymic carcinomas. Furthermore, multivariate analysis revealed that GR expression was associated with better prognosis in patients with surgically resected thymomas and thymic carcinomas. Cancer 2011;. © 2011 American Cancer Society.     

Different associations of estrogen receptor β isoforms, ERβ1 and ERβ2, expression levels with tumor size and survival in early- and late-onset breast cancer

Background

In breast cancer, little is known about the consequences of co-expression of ERα with the second estrogen receptor, ERβ, and its isoforms in light of their joint prognostic value. Previously reported correlations have been based mostly on independent ERα and ERβ expression levels in breast tumors.

Purpose

To address whether the expression ratio of ERα and ERβ and its isoforms may be a more important parameter than their absolute levels, we analyzed relative mRNA expression ratios of ERβ1 to ERβ2 and ERα in 74 clinical samples of invasive breast cancer including 39 early-onset and 35 late-onset breast cancers. Expression levels were correlated with clinical and histopathological parameters and disease-free interval.

Results

A specific correlation of ERβ1 expression levels with tumor size was detected in early-onset breast cancer patients and of ERβ2 levels with tumor size in late-onset patients. Expression of both ERβ isoforms inversely correlated with expression of the two estrogen regulated genes, progesterone receptor and pS2 in both groups. Higher levels of ERβ2 than ERβ1 isoform were associated with a better outcome in late-onset patients.

Conclusions

Our results suggest that different isoforms of ERβ may be involved in suppression of tumor growth in young and elder patients and may have different prognostic values.     

Pulse Frequency in Pulsed Brachytherapy Based on Tissue Repair Kinetics

Purpose: Investigation of normal tissue sparing in pulsed brachytherapy (PB) relative to continuous low-dose rate irradiation (CLDR) by adjusting pulse frequency based on tissue repair characteristics.Method: Using the linear quadratic model, the relative effectiveness (RE) of a 20 Gy boost was calculated for tissue with an α/β ratio ranging from 2 to 10 Gy and a half-time of sublethal damage repair between 0.1 and 3 h. The boost dose was considered to be delivered either in a number of pulses varying from 2 to 25, or continuously at a dose rate of 0.50, 0.80, or 1.20 Gy/h.Results: The RE of 20 Gy was found to be identical for PB in 25 pulses of 0.80 Gy each h and CLDR delivered at 0.80 Gy/h for any α/β value and for a repair half-time > 0.75 h. When normal tissue repair half-times are assumed to be longer than tumor repair half-times, normal tissue sparing can be obtained, within the restriction of a fixed overall treatment time, with higher dose per pulse and longer period time (time elapsed between start of pulse n and start of pulse n + 1). An optimum relative normal tissue sparing larger than 10% was found with 4 pulses of 5 Gy every 8 h. Hence, a therapeutic gain might be obtained when changing from CLDR to PB by adjusting the physical dose in such a way that the biological dose on the tumor is maintained. The normal tissue-sparing phenomenon can be explained by an increase in RE with longer period time for tissue with high α/β ratio and fast or intermediate repair half-time, and the RE for tissue with low α/β ratio and long repair half-time remains almost constant.Conclusion: Within the benchmark of the LQ model, advantage in normal tissue-sparing is expected when matching the pulse frequency to the repair kinetics of the normal tissue exposed. A period time longer than 1 h may lead to a reduction of late normal tissue complications. This theoretical advantage emphasizes the need for better knowledge of human tissue-repair kinetics.