Cellular binding proteins for vitamin A in human carcinomas and in normal tissues

Blinded analyses of the concentrations of binding proteins for retinol and retinoic acid (CRABP) in homogenates of cancer and normal tissue aliquots obtained from human cervix, endometrium, ovary, breast, and lung were carried out by the sucrose gradient ultracentrifugation technique. In carcinomas of the cervix and endometrium, CRABP mean values of 50.4 and 123.2 pmol/g tissue, respectively were detected. Such concentrations represent a 3- and 4-fold increase over the mean values of CRABP in the normal cervix (16.9 pmol/g) and normal endometrium (30.8 pmol/g), respectively. In carcinomas of the ovary, the mean CRABP level was 128.6 pmol/g compared to the maximal mean value of less than or equal to 0.46 pmol/g in the normal ovary. Elevated levels of CRABP were also found in breast and lung carcinomas compared to the amounts detected in the same patient in normal tissue aliquots of the same organ. The differences between CRABP concentrations in cervical, endometrial, ovarian, and breast carcinomas and those in normal tissue are statistically significant. In contrast, cellular retinol-binding protein concentrations were reduced in the endometrial, ovarian, breast, and lung carcinomas compared to normal tissues. There were no significant differences between the log-mean concentrations of cellular retinol-binding proteins in the cytosols from tissue aliquots of carcinoma of the cervix and those in the cytosols from tissue aliquots of normal cervix.     

Cellular binding proteins for vitamin A in the normal human uterine cervix and in dysplasias

Cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein are present in the cytosol of normal human uterine cervical tissues, as detected by ultracentrifugation analysis. Both binding proteins have characteristically high specificity for their respective ligands. In sucrose gradients, both proteins sediment in the 2S region and are of similar molecular weight (M.W. approximately 14,000). In blind analyses of cervical biopsies, obtained under direct vision by colposcopy of normal women (control) or from patients histopathologically diagnosed to have dysplasias or carcinoma in situ (study group), CRBP was not detectable by sucrose gradient analysis in 78.8% of the 33 abnormal biopsies, compared to 23.5% of the 34 controls. This difference was statistically significant (p less than 0.005). In biopsies in which CRBP was detected, the mean levels were 2.76 and 0.72 pmol/mg protein in the cytosol for the control and study groups, respectively. In some subjects from each group, cellular retinoic acid-binding protein but not CRBP was detected in the biopsied tissue. The presence and role of these binding proteins in vitamin A metabolism, epithelial maturation and differentiation in cervical dysplasias, and in situ lesions remain to be investigated.     

Cellular vitamin A-binding proteins in liver tumors and adjacent tissues

The levels of cellular retinol-binding protein (CRBP) and retinoic acid-binding protein (CRABP) were assessed in surgically resected ten human liver tumors and their adjacent tissues by sucrose density gradient centrifugation and Scatchard analysis. The tissues adjacent to hepatocellular carcinomas usually showed portal cirrhosis or hepatitis. There was no significant difference in the dissociation constant (Kd) values of binding proteins of CRBP or CRABP between tumors and adjacent tissues. Five of the ten liver tumors showed similar levels of CRBP as in their adjacent tissues whereas three of the ten liver tumors showed about 40% or less contents of CRBP in comparison with their adjacent tissues and two others had no CRBP activity. By contrast, CRABP activities were found in five liver tumors and in adjacent tissues of one case, whereas there were no detectable CRABP activities in the adjacent tissues except in one case.     

Specific binding proteins for selenium in rat tissues

The preventive and therapeutic potential of selenium (Se), amicronutrient, against cancer has been well documented in severaltest systems, but the mechanism of its action is not known.The possibility that Se might function in a manner similar tosteroid hormones and retinoids through mediation of cellularreceptors was examined. A specific 2S cellular binding protein(SeBP) for Na₂[⁷⁵Se]O₃ was detected in rat tissue extracts.Liver and intestine exhibited highest levels of SeBP, and heart,uterus and spleen had the lowest levels. Oral administrationof Na₂[⁷⁵Se]O₃ to rats resulted in its uptake by the tissueswith concomitant appearance of [⁷⁵Se]SeBP complex. The proteinbinds sodium selenite with moderately high affinity; the apparentdissociation constant was determined by Scatchard analysis tobe 1.1x10^–7 M. SeBP focused at pH 5.3 upon isoelectricfocusing in ampholines of pH 3–10. Competitive bindingaffinity studies with unlabeled test compounds revealed thatselenium dioxide and selenocystine showed high binding affinity(90–95%) for the selenite-binding site on SeBP. Sodiumselenate, elemental Se powder, and selenomethionine, however,showed poor competition with sodium selenite. Biological activityof the above selenocompounds, as expressed by others, correlatewith their binding affinities for SeBP. Sodium sulfite showed35% inhibition of Na₂[⁷⁵Se]O₃ binding, but sulfate showed none.Two ultimate carcinogens, N-methyl-N-nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine,and two retinoids, retinol and retinoic acid, showed <10%inhibition of binding. Interaction of Se with SeBP is completelyblocked by thiol inhibitors. Plasma transport of Na₂[⁷⁵Se]O₃is mediated by a protein with a mol. wt of 68000, which is presentlyidentified, by immunoprecipitation studies as well as by Affi-GelBlue column chromatographic experiments, as serum albumin. Theresults suggest that the plasma transport of Se is facilitatedby albumin, and that the intracellular transport of Se for itsbiological functions is accomplished by SeBP.     

Mucins and mucin binding proteins in colorectal cancer

Mucins are high-molecular weight epithelial glycoproteins with a high content of clustered oligosaccharides O-glycosidically linked to tandem repeat peptides rich in threonine, serine, and proline. There are two structurally and functionally distinct classes of mucins: secreted gel-forming mucins (MUC2, MUC5AC, MUC5B, and MUC6) and transmembrane mucins (MUC1, MUC3A, MUC3B, MUC4, MUC12, MUC17), although the products of some MUC genes do not fit well into either class (MUC7, MUC8, MUC9, MUC13, MUC15, MUC16).MUC1 mucin, as detected immunologically, is increased in expression in colon cancers, which correlates with a worse prognosis. Expression of MUC2 secreted gel-forming mucin is generally decreased in colorectal adenocarcinoma, but preserved in mucinous carcinomas, a distinct subtype of colon cancer associated with microsatellite instability. Another secreted gel-forming mucin, MUC5AC, a product of normal gastric mucosa, is absent from normal colon, but frequently present in colorectal adenomas and colon cancers.The O-glycosidically linked oligosaccharides of mucins can be described in terms of core type, backbone type, and peripheral structures. Colon cancer mucins have differences in both core carbohydrates and in peripheral carbohydrate structures that are being investigated as diagnostic and prognostic markers, and also as targets for cancer vaccines. Colon cancer mucins typically have increases in three core structures: Tn antigen (GalNAcThr/Ser), TF antigen (Gal3GalNAc) and sialyl Tn (NeuAc6GalNAc). The type 3 core (GlcNAc3GalNAc) predominant in normal colonic mucin is lacking in colon cancer mucins. There are cancer-associated alterations in the peripheral carbohydrates of colonic mucins including a decrease in O-acetyl-sialic acid and a decrease in sulfation. There are, however, cancer-associated increases in sialyl LeX and related structures on mucins and other glycoproteins that can serve as ligands for selectins, increasing the metastatic capacity of colon cancer cells. The endogenous galactoside-binding protein galectin-3, which is expressed at higher levels in colon cancers than normal colon, binds to colon cancer mucin as well as other glycoproteins. Interference of the binding of selectins and galectin-3 to mucin may show therapeutic or preventative promise for colon cancer.     

Lectin resistance and lectin binding in a rat adenocarcinoma

Cell cultures from the R3230 AC rat mammary adenocarcinoma, when injected i.v. into F 344 rats, invariably produce multiple lung foci within 10 days. We compared the colonization potential of cultures exposed to 100 micrograms/ml medium of both Concanavalin A and Wheat Germ Agglutinin for 5 passages with the original cell line. All cell lines retained their ability to bind both lectins and to grow after subcutaneous implant; however, the lectin resistant after subcutaneous implant; however, the lectin resistant variant was found to have completely lost its capacity to nidate in the lung.     

Cellular differentiation of rat uterine adenocarcinoma cells by progesterone in vitro.

Transplantable cloned HTP/Cl culture was a stable line derived from a rat uterine adenocarcinoma that was induced by 7,12-dimethylbenz(a)anthracene in vivo and did not display density-dependent inhibition of growth. This HTP/Cl culture easily adapted to grow in a culture medium containing progesterone, 8 mug/ml. As compared with HTP/Cl culture, HTP/Cl/P8 culture grown in the presence of progesterone was contact inhibited, and a cellular differentiation was observed in the tumor tissues that developed after inoculation of cells. These actions of progesterone on uterine adenocarcinoma cells were completely reversible on removal of the hormone in vitro. These results appeared to indicate that progesterone was involved in the regulation of both cellular proliferation and differentiation. The possible mechanisms of the regulation of rat uterine adenocarcinoma cells by progesterone are discussed in relation to cellular levels of cyclic adenosine 3':5'-monophosphate in vitro.     

Retinoic acid receptors and retinoid binding proteins in endometrial adenocarcinoma: Differential expression of cellular retinoid binding proteins in endometrioid tumours

Retinoic acid is apparently required for the normal differentiation of reproductive epithelium. Cellular abnormalities in retinoid homeostasis could be a factor in the development of endometrial malignancy. We have thus investigated the expression of nuclear retinoic acid receptors (RARs and RXRs) and cellular binding proteins for retinol (CRBP) and retinoic acid (CRABP) in endometrial adenocarcinoma of the endometrioid histological subtype. Ten grade 1, 11 grade 2 and 10 grade 3 tumour samples, as well as 4 samples of severe atypical precan-cerous endometrial hyperplasia, were studied. No significant difference in expression of RAR-β was detected in tumour samples compared with normal epithelial cells. RAR-γ was significantly elevated in grade 1 and 2 carcinomas, but this may be due to greater stromal cell involvement in these lower grade tumours. There was significant elevation of CRBP 1 mRNA in tumour samples. Furthermore, although undetectable in normal endometrial epithelium, CRABP 1 was expressed in 3/11 grade 2 and 9/10 grade 3 carcinomas, with expression being significantly higher where the primary tumour had invaded more than 50% of the total myometrial thickness. Analysis of 2 epithelial-like endometrial adenocarcinoma cell lines supported the idea that CRABP 1 expression is characteristic of poorly differentiated endometrial adenocarcinoma. Our data suggest that alterations in mechanisms of retinoid homeostasis are a feature of endometrial adenocarcinoma and may contribute to the severity of disease. © 1995 Wiley-Liss, Inc.     

Bcl-w expression in colorectal adenocarcinoma

We have found that the anti-apoptotic Bcl-2 family protein, Bcl-w, was frequently expressed in colorectal adenocarcinomas, with 69/75 showing positive staining with anti-Bcl-w IgG. Adenomas demonstrated a much lower frequency of Bcl-w expression (only 1 of 17), as did adenocarcinomas from other epithelial tissues such as breast (0/8), stomach (1112) and cervix (0/12). Bcl-w status could be related to the histopathological classification of the tumours, with TNM stage III tumours showing significantly higher levels of expression than tumours of better prognostic grade (at P = 0.009). Those patients with node involvement also had tumours with significantly elevated levels of Bcl-w (at P = 0.02), compared to those which were node-negative. The results suggest that Bcl-w could play a general role in the progression from adenoma to adenocarcinoma in the colorectal epithelium. Currently, more data are being collected to allow us to assess the importance of Bcl-w for disease progression and patient survival.     

人大肠腺瘤和腺癌之间相关差异蛋白的蛋白质组学研究

目的　应用蛋白质组学的方法,建立人大肠腺癌组织、大肠腺瘤组织、正常大肠黏膜组织的二维电泳图谱,筛选并鉴定三者之间的差异蛋白,发现人大肠癌的潜在生物学标志物。方法　收集同时患有大肠腺瘤的大肠腺癌患者１０例,术前及术后病理证实均为Ｄｕｋｅｓ’Ｂ期。取同一患者术后大肠癌组织、距大肠癌组织１５ｃｍ以上正常大肠黏膜组织以及肠镜下切除大肠腺瘤组织,提取不同组织总蛋白进行二维电泳,用ＩｍａｇｅＭａｓｔｅｒ２ＤＰｌａｔｉｎｕｍ５.０凝胶图像分析软件对双向电泳图依次进行凝胶点的检测、背景消减和编号。识别不同组织之间的差异蛋白点,对差异蛋白点用胶内酶解后行质谱分析和网上数据库鉴定。结果　大肠腺癌组织、大肠腺瘤组织和正常大肠黏膜组织之间存在９０个差异蛋白点。对这些差异点进行质谱分析,经数据库鉴定出７１个蛋白质。经分析,大肠腺瘤组织与正常大肠黏膜组织相比,１７个蛋白表达上调,９个蛋白表达下调;大肠腺癌组织与正常大肠黏膜组织比较,４７个蛋白表达上调,１２个蛋白表达下调;大肠腺癌组织与大肠腺瘤组织相比,３５个蛋白表达上调,８个蛋白表达下调。结论　大肠腺癌组织、大肠腺瘤组织和正常大肠黏膜组织之间存在差异蛋白表达,可能与大肠腺癌的发生发展有关;应用蛋白质组学方法筛选人大肠腺癌发生发展相关蛋白是可行的。     

bcl-2和P53基因蛋白在大肠腺瘤及腺癌的表达及其意义

目的　探讨bcl 2和P5 3基因蛋白在大肠癌发生中的作用和意义及其与大肠癌分级分期的关系。方法　采用免疫组织化学方法 ,检测bcl 2和P5 3蛋白在 2 6例大肠腺瘤和 12 5例大肠腺癌中的表达。结果　bcl 2蛋白在大肠腺瘤的阳性表达率为 76.9% ( 2 0 /2 6) ,在大肠腺癌为 65 .6% ( 82 /12 5 ) ,其阳性率有随肿瘤分化程度降低而随之下降的趋势 (P <0 .0 5 ) ,bcl 2蛋白阳性率随大肠癌分期的增高而下降 (P <0 .0 5 )。P5 3蛋白仅在 1例重度非典型增生的大肠腺瘤有阳性表达 ( 3 .8% ) ,而在大肠腺癌的阳性表达率达 80 % ( 10 0 /12 5 ) ,但其阳性率与肿瘤的分化程度和肿瘤分期无递增递减规律。 46.4% ( 5 8/12 5 )的腺癌有bcl 2和P5 3蛋白的共同表达。两种蛋白的阳性表达率存在负相关 (rs =-0 .68,P <0 .0 5 )。结论　bcl 2基因在大肠癌发生发展的早期阶段起作用 ,而P5 3蛋白的过表达可能在大肠癌发生发展的晚期阶段。bcl 2和P5 3基因蛋白的联合检测可以作为临床早期判断大肠腺瘤癌变的一个重要参考指标     

Endobronchial metastases in colorectal adenocarcinoma.

Between 1982 and 1990, 2388 bronchoscopic examinations were carried out in patients with cancer in our hospital. A diagnosis of endobronchial metastasis was established in 30 patients (2.09%), with the following primary tumors in descending order of frequency: breast, large bowel, melanoma, neuroblastoma, leiomyosarcoma and endometrial. Despite the rarity of endobronchial metastases secondary to colon adenocarcinoma, we were able to study 3 cases from our Center. In one case the diagnosis of endobronchial metastasis was simultaneous with that of the primary tumor, and in the other 2 this metastatic complication occurred 16 and 42 months, after the original diagnosis. When this complication occurred, the stage of the disease was advanced in all 3 cases: 2 were Dukes' stage C and one stage D. Although this metastatic location usually implies a very negative prognosis as regards life expectancy, it did not seem to significantly reduce the latter in our patients.     

set基因在人结直肠腺癌中表达的研究

目的探究set基因在人类结直肠腺癌组织中的表达情况及其表达水平与临床病理特征之间的关系。方法 通过半定量PCR检测set基因在39例结直肠腺癌组织与自身对照的正常结直肠组织中的mRNA表达,免疫组织化学检测SET蛋白在9例结直肠腺癌患者癌组织及其自身对照的正常结直肠组织中的表达。结果 (1)set基因在26例结直肠腺癌组织中的mRNA表达水平高于其自身对照正常结直肠组织,占总数的66.7%的,其中6例患者set基因在结直肠腺癌组织中的表达水平是其在正常癌旁组织中表达水平的2倍以上,最高达22倍(P<0.05)。(2)免疫组织化学显示,SET蛋白在结肠腺癌和癌旁组织细胞核中均表达,其中8例患者的癌组织SET蛋白表达水平明显高于癌旁组织(P<0.05)。结论 set基因在结肠癌中的表达上调,可能与结直肠腺癌发病有关。     

Expression of Id helix-loop-helix proteins in colorectal adenocarcinoma correlates with p53 expression and mitotic index.

Id helix-loop-helix (HLH) proteins function as regulators of cell growth and differentiation and when overexpressed can induce malignant transformation. In a series of 34 cases of primary human colorectal adenocarcinoma, immunoreactivity for Id1, Id2, and Id3 was found to be significantly elevated in tumor compared with normal mucosa (P = 0.001 for Id1 and Id2; P = 0.002 for Id3). No elevation of Id expression was observed in 17 cases of adenoma. Expression of Id1 and to a lesser extent of Id2 was correlated with mitotic index (P = 0.005 for Id1; P = 0.042 for Id2) in human adenocarcinomas, and expression of all three Id proteins was correlated with p53 immunoreactivity (a marker of mutational 'inactivation' of p53 function; P = 0.002 for Id1; P = 0.006 for Id2; P = 0.016 for Id3). In normal intestinal mucosa of p53-null mice and in spontaneous tumors arising in Min+/- mice, expression of all three Id proteins was also found to be up-regulated. Antisense oligonucleotide blockade of Id protein expression inhibited the proliferation of human adenocarcinoma cells. Enforced, ectopic expression of the E47 basic HLH (bHLH) protein in human adenocarcinoma cell lines efficiently sequestered endogenous Id proteins as Id-bHLH heterodimers, as shown by coimmunoprecipitation and subcellular colocalization studies. This led to growth arrest of the cells. Enforced overexpression of a mutant E47 protein, deficient in transactivation and DNA binding function, also partially inhibited cell growth. Taken together, these data imply that deregulated expression of Id proteins in colorectal adenocarcinoma arises at least in part as a consequence of loss of p53 function and contributes to the uncontrolled proliferation of tumor cells in colorectal cancer.     

A case of heterogeneous sensitivity to panitumumab in cetuximab-refractory colorectal adenocarcinoma metastases

We present the case of a 43-year-old-man with wild-type KRAS and BRAF colorectal adenocarcinoma that was metastatic to the liver and lung. The patient initially received neoadjuvant chemotherapy with FOLFOX and bevacizumab, followed by surgical resection of the primary tumor and hepatic metastases. His disease recurred shortly after surgery and he was treated with FOLFIRI plus the anti-EGFR antibody cetuximab. After this regimen failed to arrest his disease progression, treatment with FOLFIRI in combination with another anti-EGFR antibody, panitumumab was started. While on this therapy, the patient''s lung nodules remained largely stable but metastatic lesions within the liver continued to progress. Our case highlights the differences between panitumumab and cetuximab, and contemplates the possible explanations for this patient''s apparently heterogeneous disease progression within the liver despite stabilization of multiple pulmonary nodules.