Retinoic acid-binding protein in human breast cancer and dysplasia.

Seventy-five specimens of human breast tissue were checked for the presence of cellular retinoic acid-binding protein (cRABP). Fifty-two percent of the primary carcinomas and 43% of the dysplastic breast lesions (stage MII) contained detectable amounts of crabp, whereas no cRABP was found in normal tissue. Sucrose gradient centrifugation and electrophoresis on agarose were used for analysis of the presence of cRABP. The cRABP of human origin (normal uterus and neoplastic mammary tissue) differed in its mobility in agarose electrophoresis from that of rat testis cRABP.     

Correlation between retinoid inhibition of N-methyl-N-nitrosourea-induced mammary carcinogenesis and levels of retinoic acid binding proteins

A correlation was made between the ability of retinoids to suppressN-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesisand the levels of cytosolic retinoic acid binding proteins (cRABP)in the cytosol of MNU-induced mammary tumors. Although retinylacetate and N-(4-hydroxyphenyl)retinamide were found to be effectiveinhibitors of mammary carcinogenesis in intact hosts, both retinoidswere significantly more active in ovariectomized rats than inintact animals. Quantitative analyses of cRABP in the tumorsindicated that mammary cancers arising in animals which wereovariectomized one week after MNU administration contained significantlyincreased concentrations of cRABP compared to cancers appearingin intact rats. In addition, when animals bearing palpable mammarytumors were ovariectomized, the tumors which continued to growcontained significantly higher levels of cRABP than did tumorswhich stopped growing or regressed. These data suggest thatthe selective inhibition of ovarian hormone-independent mammarycancer by retinyl acetate and N-(4-hydroxyphenyl)retinamidemay be mediated through an increased level of cRABP in tumorcells of ovariectomized hosts.     

Cellular binding proteins for vitamin A in colorectal adenocarcinoma of rat

Rat colorectal mucosa was examined during the course of carcinogenesis, induced by chronic administration of 1,2-dimethylhydrazine (DMH), for the presence and amount of cellular retinol-binding protein (CRBP) and cellular retinoic acid-binding protein. These two binding proteins are implicated in the action of vitamin A in normal and neoplastic tissue. Induced adenocarcinomas were found to contain low levels of cellular retinoic acid-binding protein (10 pmol/g), similar to the levels found in adjacent mucosa of the same animal and also in colorectal mucosa from normal rats or rats chronically treated with DMH. However, the adenocarcinomas had high levels of CRBP (300 to 500 pmol/g), and these levels were dramatically higher than levels of CRBP in adjacent mucosa of the same animal (40 to 100 pmol/g), colorectal mucosa from normal rats (20 pmol/g), or colorectal mucosa from rats chronically treated with DMH (22 to 25 pmol/g). Consequently, the increase in CRBP occurred only with tumor appearance and not with the general hyperplasia of the crypts caused by DMH administration. The CRBP of the tumor was associated with endogenous retinol (77 to 100% saturation) and was similar to, if not identical with, CRBP of normal tissue, as judged by fluorescence spectra, sedimentation behavior, and elution position on Sephadex G-75.     

The Effect of Sodium Butyrate and Retinoic Acid on Growth and CEA Production in a Series of Human Colorectal Tumor Cell Lines Representing Different States of Differentiation

The effect of sodium butyrate and retinoic acid added singly or in combination on substrate-dependent growth, colonization efficiency in soft agar, and carcinoembryonic antigen (CEA) production in three human colorectal carcinoma cell lines differing in their degree of differentiation was studied. All three colon cancer cell lines regardless of their state of differentiation had their growth markedly slowed by sodium butyrate, and to a lesser extent by retinoic acid. When both agents were added together, a small synergistic inhibition of growth was noted in all the cell lines. Butyrate eliminated colony formation in soft agar in all three cell lines, however, retinoic acid only reduced colony formation in the well differentiated cell line DLD-2. Sodium butyrate was able to induce CEA production in the undifferentiated cell (MIP-101) and the moderately differentiated cells (clone D) which were previously negative for this marker. It also enhanced the baseline production of CEA in the well differentiated cells (DLD-2). Retinoic acid did not induce CEA production in clone D or MIP-101 cells, but did enhance the production of CEA in DLD-2 cells. When both retinoic acid and sodium butyrate were added together, CEA production was either additive (DLD-2) or was inhibited (clone D and MIP-101). One explanation of these results is that only well differentiated cells have functional cellular retinoic acid-binding protein (cRABP), and that certain actions of retinoic acid (inhibition of anchorage-dependent growth) are independent of the presence of cRABP.     

Retinoid receptors in epidermoid carcinoma of the ORL sphere

Head and neck tumors from 20 patients were investigated for the presence and the level of retinoid binding proteins (cellular retinoic acid binding protein or CRABP, cellular retinol binding protein or CRBP). A binding assay-gel filtration technic was used. CRABP were found in all tumors and in the adjacent normal tissues. A decreased level was associated with poor prognostic factors. CRBP were only found in some tumors and were absent from the adjacent normal tissue and from benign tumors. These results are discussed as regards their interest in prognostic evaluation and therapeutic orientation.     

Biochemical markers in colon tumorigenesis: retinoic acid and dihydrotestosterone-binding proteins

A specific retinoic acid-binding protein (RABP) and a dihydrotestosterone-binding protein (DHTBP) appeared in colon tumors as well as in tissues surrounding the the tumors, but the proteins were nondetectable in normal adult human colon tissues. We have analyzed a total of 105 human colon tumors and related specimens for the presence of RABP and DHTBP as possible biochemical markers in colon tumorigenesis. The tissue or tumor extracts after incubation with (3H) retinoic acid or (3H) dihydrotestosterone were sedimented on sucrose gradients, and the binding proteins were detected from the 2S (RABP) or 6-7S (DHTBP) radioactive peaks. The overall results of the analysis illustrate that about 78% of the 74 human colon, rectum, cecum, and colorectum tumors analyzed contained RABP in detectable amounts. Thirty percent of the 20 colon tissues isolated from subjects suspected for colon cancer and 18% of the 11 normal colon tissues from autopsies contained detectable amounts of the binding protein. A comparative study on the quantitative amounts of RABP and DHTBP in colon tumors and related tissues indicates that the amount of retinoic acid or dihydrotestosterone bound per mg protein ranged from 0.8 to 5.1 pmoles in the binding protein-positive specimens. However, the relative amounts of the two binding proteins in these tissues were not in the same proportions. Appearance of RABP and/or DHTBP in the surrounding tissues of colon tumors correlated with the amounts of the binding protein(s) in the tumors. RABP of human colon tumor shared the same ligand specificity and other physicochemical characteristics as RABP of other species.     

Protein kinase A regulatory subunits in colon cancer

The protein kinase A (PKA) is classified as type I or II depending on the association of the catalytic subunit with either the R(I) or R(II) regulatory subunits. Alterations in the levels of these regulatory subunits and PKA activity itself appear to affect cellular proliferation and tumorigenesis. We examined colorectal tumor specimens from 45 patients to investigate the potential role of cAMP-related signaling molecules in regulating tumorigenesis. Western blot analysis (PKA subunit protein levels) and in vitro kemptide phosphorylation assays (PKA catalytic subunit activity) were performed on human colorectal tumor tissue homogenates. R(I)beta protein levels were decreased 200% in ascending and 50% in descending colonic tumors compared to adjacent mucosa. R(II) protein levels were decreased 77% in descending colonic tumors but no change was observed in ascending colonic tumors compared to adjacent mucosa. PKA activity and the absolute amount of catalytic subunit protein in ascending and descending tumors were unchanged compared to adjacent mucosa. Differences in cAMP-related signaling molecules exist between neoplastic and normal colorectal tissues. These differences may not only serve as potential therapeutic targets for chemotherapeutic agents, but also lead to the identification of novel regulatory mechanisms involved in cellular proliferation and tumorigenesis.     

Presence of free retinol receptor (cRBP) and retinoic acid receptor (cRABP) in human skin tumors

The concentrations of cellular retinoic acid binding protein (cRABP) and free cellular retinol binding protein (cRBP) were determined by ultracentrifugation in sucrose gradient in the cytosol of 41 human skin tumours (14 melanomas, 19 basal cell carcinomas, 8 squamous cell carcinomas). cRBP was found respectively in 36%, 42% and 37% of the studied samples. On the contrary, cRABP was more frequently found in carcinomas (89% in basal cell carcinomas and 100% in squamous cell carcinomas) than in melanomas (21%) (p less than 0.001). These results are discussed according to the different embryologic origin of carcinomas and melanomas. Furthermore, the better efficiency of synthetic retinoids in carcinomas than in melanomas should be explained by a different way of action in these 2 kinds of tumours.     

Differential immunohistochemical detection of amphiregulin and cripto in human normal colon and colorectal tumors.

Thirty-six primary human colorectal tumors, 43 noninvolved colon samples that were adjacent to either carcinomas of adenomas, 22 adenomas, and nine normal colon specimens were immunohistochemically examined for the presence and localization of two epidermal growth factor-related peptides, amphiregulin (AR) and cripto. Within the primary tumors, 18 (50%) showed moderate levels of AR expression. Approximately 60% of the tubular and tubulovillous adenomas were positive for AR expression, whereas only 15% of the adjacent, noninvolved colon mucosa expressed AR. A greater proportion of well-differentiated tumors (71%) were positive for AR expression than were poorly differentiated tumors (18%). All of the nine normal colon specimens were positive. Consequently, AR expression appeared to be associated with both normal and malignant epithelial cells that were more differentiated. The distribution of cripto expression was different. Seventy-nine % of the colon tumors expressed cripto with a frequency of expression that was approximately equivalent between well-differentiated and poorly differentiated tumors. Approximately 86% of the tubulovillous adenomas, but only 43% of the tubular adenomas, were positive for cripto expression. In contrast, whereas AR was expressed in normal colon specimens, none of these tissues expressed cripto, and only 12% of the noninvolved normal colon samples adjacent to tumors or adenomas were positive for cripto. Cripto expression therefore appeared related to neoplasia. These data suggest that AR and cripto may be functioning as potential autocrine and/or paracrine growth factors in the colon and that the differential expression of cripto may serve as a potential tumor marker for colonic carcinogenesis.     

Evidence of p53 immunohistochemical overexpression in ethmoidal mucosa of woodworkers

A high risk of neoplastic transformation of nasal and paranasal sinuses mucosa is related to the occupational exposure to wood dust, however no conclusive data have been reported up to now about morphological precursors of these tumors, mechanisms of carcinogenesis and role of p53 gene. Immunohistochemical overexpression of protein p53 (DO7 clone) by epithelial cells of ethmoidal mucosa was investigated on 60 woodworkers occupationally exposed for a minimum of 10 years, on 50 functional and/or esthetic nasal surgery patients (control group) and on 15 cases of intestinal-type adenocarcinoma, 10 of these involving subjects who had a longtime exposure to wood dust. In almost all the woodworkers (92%) the normal ciliated epithelium showed tracts of squamous metaplasia. The mean percentage of p53-positive cells in woodworkers and in controls was 28.6 and 7.97%, respectively, in metaplastic epithelium (P<0.001), 11.7 and 2.08% in ciliated epithelium (P<0.001), 12.46 and 1.03% (P<0.001) in the sero-mucous glands of the nasal stroma. Both in tracts of metaplastic epithelium and in those of ciliated epithelium, positive cells were distributed in basal and suprabasal layers. A high number of p53-positive cells was also observed in the normal ciliated epithelium close to the neoplastic cells, of intestinal-type adenocarcinomas affecting subjects with longtime exposure to wood dust. Moreover, a higher number of p53 positive neoplastic cells was showed by the cases occurring in occupationally exposed patients than by the others. The following conclusions can be drawn: (1) in the ethmoidal mucosa, a region at high risk of carcinogenesis in subjects exposed to wood dust, epithelial cells overexpress p53 protein, and this may be linked to the presence of p53 gene mutations; malignant transformation, as at other head and neck sites, may thus occur through this pathway; (2) the presence of p53 overexpression in sero-mucous glands is in keeping with the histogenesis of some tumors from these structures; (3) since tumors of nose and paranasal sinuses, mainly adenocarcinoma, are recognized as occupational neoplasias, immunohistochemical evaluation of p53, perhaps combined with molecular methods, could be the first step to detect subjects at high risk of carcinogenesis among woodworkers.     

Ornithine decarboxylase activity and polyamine content in adenocarcinomas of human stomach and large intestine

Ornithine decarboxylase (ODC) activity and differential polyamine composition have been studied in macroscopically normal mucosa, adjacent mucosa, and neoplastic tissue of 95 patients with adenocarcinomas of stomach and large intestine. In tumors, we found increased ODC activity and polyamine content as compared with surrounding mucosa. ODC activity in macroscopically normal tissue of patients with tumors of stomach and large intestine increased as the disease progressed. An inverse relationship was observed between ODC activity in adenocarcinomas and differentiation.     

MRP和P—gp与食管鳞癌发生发展的关系研究

目的探讨MRP、P-gP在食管鳞癌中的表达情况及与食管鳞癌发生发展的关系。方法采用免疫组织化学sP法研究了49例食管鳞癌组织中MRP和P-gp的表达情况,探讨二者与肿瘤发生发展的关系。结果49例食管鳞癌癌组织、癌旁非典型增生组织、正常食管黏膜组织MRP阳性表达率分别为63．3％（31／49）、40．0％（12／30）和20．4％（10／49）。癌组织中MRP阳性表达率与相应的癌旁组织、正常食管黏膜组织比较,差异有显著性（P〈0．05）。49例食管鳞癌癌组织、癌旁非典型增生组织、正常食管黏膜组织中P-gP阳性表达率分别为61．2％（30／49）、36．7％（11／30）和18．4％（9／49）。癌组织中P-gp阳性表达率与相应的癌旁组织、正常食管黏膜组织比较,差异有显著性（P〈0．05）。结论食管鳞癌组织、癌旁不典型增生组织及正常黏膜组织中MRP和P—gP阳性表达率均依次降低,并且癌组织与癌旁不典型增生组织和正常黏膜组织中MRP和P—gP阳性表达间相比,差异有显著性,表明MRP和P-gP参与食管鳞癌的发生发展过程,可作为食管上皮细胞恶性转化的标志。     

Cytoplasmic retinoic acid-binding protein in retinoic acid-resistant human breast cancer sublines

Two sublines resistant to the growth-inhibitory effects of retinoic acid (RA) have been isolated from the parental Hs578T wild-type (W.T.) human breast cancer cell line. These sublines (Hs578T-R-1 and Hs578T-R-2) have been growing normally in 10 microM RA during more than 18 months, and their RA-resistant phenotype has remained stable after the removal of RA. The resistance is specific for RA, since their growth is still inhibited by retinol. The intracellular incorporation of [3H]RA is not deficient in the RA-resistant sublines. Cytoplasmic RA-binding protein (cRABP) is present in Hs578T-R-1 and in Hs578T-R-2 and is not different in terms of maximum binding capacity or binding affinity from cRABP in Hs578T (W.T.). These results indicate that RA resistance in these sublines is not secondary to a defect of RA uptake or of binding of RA to cRABP; the resistance may result from a defect distal to binding to cRABP, or alternatively, cRABP may not mediate this effect of RA.     

Retinoic acid-binding protein in experimental tumors and in tissues with metastatic tumor foci

Screening for retinoic acid-binding protein (RABP) in experimental tumors revealed the presence of this protein in three mammary tumors, two metastatic colon tumors, B16 melanoma. Lewis lung carcinoma, Ridgway osteogenic sarcoma, and keratoacanthoma. RABP was below the limits of detection in two weakly metastatic colon tumors and in Sarcoma 180. After s.c. implantation of RABP-containing tumors into mice, this protein could be traced in the lungs due to pulmonary metastasis. Following implantation of Lewis lung tumors, RABP was detected in the lung on the 6th day. On the 15th day after implantation, RABP was present in lung and brain, but not in other tissues where this protein was normally lacking. In primary cultures of Lewis lung carcinoma, the lower limit for detection of RABP by sucrose gradient sedimentation technique corresponded to 0.12 mg protein that was extractable from 3 X 10(5) cells. Both chick embryo skin and rabbit ear skin extracts contained RABP; the level of cellular retinol-binding protein was high in chick embryo skin but only marginal in rabbit ear. The amounts of these proteins on chick embryo skin and rabbit ear skin correlate with the biological potency of retinol and retinoic acid, as observed by others.     

利用组织芯片技术研究胃癌及其癌前病变中Bax、p53、Survivin表达的关系及意义

Objective： To explore the relationship between expressions of apoptosis-related protein Bax, Survivin and p53 and the molecular mechanisms of carcinogenesis and progression of gastric carcinoma. Methods： Tissue microarray and immunohistochemistry were used in this study. Results： The positive rate of Bax protein in gastric cancer （17.7%, 17/96） was significantly lower than those in adjacent normal mucosa （51%）, intestinal metaplasia （69.2%） and dysplasia （75%）, P 〈 0.01. The positive rate of Survivin expression in gastric cancer （80.6%, 89/98） was significantly higher than that in adjacent normal mucosa （3.9%）, P 〈 0.01. The positive rates of Survivin expression in tumors with different organ metastases （in lymph node metastasis 86.2%, liver 100% and ovarian 100%） were statistically higher than in tumors without metastasis （64.3%）, P 〈 0.05. Bax expression was correlated with Survivin but not with rap53 that was closely related to Survivin expression （P 〈 0.05） in gastric cancer. Conclusion： The abnormal expressions of Bax, Survivin and rap53 were correlated with the tumorigenesis and progression of gastric carcinoma. P53 and Survivin genes may share the similar mechanism in regulating cell apoptosis, and because of the mutation, p53 gene may lower its down-regulation to Survivin expression.     

Properties of ornithine decarboxylase in human colorectal adenocarcinomas

Ornithine decarboxylase (ODC) activity was measured in colon adenocarcinomas and adjacent normal-appearing colon mucosa from a total of 40 patients undergoing surgical resections. The enzyme activity was measured in the presence and absence of GTP, since recent work has demonstrated a GTP-activatable form of ODC in some murine and human tumors. In general, ODC specific activity was higher in adenocarcinomas than in adjacent normal-appearing mucosa. Of greater interest, however, was the finding that 13 of 40 tumors and 3 of 40 mucosae contained a GTP-activatable form of ODC. These are minimal estimates of the proportion of tissues positive for this enzyme form, since a multiple sampling protocol indicated that expression of a GTP-activatable ODC was not uniform throughout a given tumor. Chromatographic analyses of tumor extracts revealed the presence in some tumors of multiple size forms of ODC, only some of which were activated by GTP. Enzyme kinetic data indicated that the multiple forms of ODC can have different affinities for L-ornithine and that GTP can "normalize" the aberrant kinetic properties of these forms. While there was no statistically significant correlation of the presence of a GTP-activatable ODC with stage of disease, analysis of our data revealed a positive association of a GTP-activatable ODC with tumor site; a much higher percentage of tumors of the cecum contained this ODC isoform than tumors of other colonic segments (64% versus less than or equal to 25% for other sites). These results demonstrate (a) the presence of a functionally distinct form of ODC in some human colon adenocarcinomas and (b) a distinct regional distribution of this ODC form within the colon. We suggest this alteration in a key enzyme in the growth-associated pathway of polyamine biosynthesis may play a role in colon tumor progression.     

Immunoreaction at 43 kDa with Anti-ubiquitin Antibody in Breast Neoplasms

Protein ubiquitination has been implicated in ATP-dependent protein turnover and normal cell proliferation. To investigate whether the ubiqnitin-mediated system is functionally involved in the cancerous state, we examined changes in protein ubiquitination in 52 surgically resected primary breast tumors. Imnmnohistochcmically, ubiquitin (Ub) was identified in the cytoplasm of cancer cells, which were stained more strongly than adjacent normal ductal epithelium. Corresponding immunoblot analysis of normal and neoplastic regions of human breast showed that the immunoreaction for Ub at about 43 kDa was increased in all of the tumors (100%), regardless of the clinical stage or histologic grade. This protein, which gave a single spot on two-dimensional gel electrophoresis, had partial amino acid sequences which were identical to those of actin family members. Our results suggest that ubiquitination of this 43-kDa protein may be involved in the carcinogenesis or biological characteristics of human breast neoplasms.     

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.     

Expression of nuclear retinoid receptors in normal, premalignant and malignant gastric tissues determined by in situ hybridization.

Retinoids exhibit multiple functions through interaction with nuclear retinoid receptors and have growth-suppressive activity on gastric cancer cells. To better understand the roles of nuclear retinoid receptors during gastric carcinogenesis, we have used in situ hybridization to investigate expression of retinoic acid receptors (RARs) and retinoid x receptors (RXRs) in premalignant and malignant formalin-fixed paraffin-embedded gastric tissues. Histological sections of eight normal, 17 distal normal and nine gastric cancer tissues were hybridized with non-radioactive RNA probes for subtypes of RAR and RXR. Expression of RAR alpha, RAR beta, RAR gamma, RXR alpha and RXR beta was found in most cell types in gastric mucosa tissues from normal individuals as well as in distal normal tissues from cancer patients. Expression of RAR alpha and RAR beta were found in three and seven cancer tissues, respectively, and levels of RXR alpha mRNA were significantly decreased in poorly differentiated cancer tissues. Among the five investigated nuclear retinoid receptors, only expression of RAR alpha mRNA was significantly decreased in intestinal metaplasia, dysplasia and cancer tissues when compared to adjacent normal tissues. In conclusion, normal gastric mucosa expressed both RARs and RXRs, which supports the physiological role of retinoic acid on normal gastric mucosa. The decrease in RAR alpha expression in premalignant and malignant gastric tissues suggests a significant role of RAR alpha during gastric carcinogenesis.     

大肠癌细胞转铁蛋白受体表达的临床意义

应用抗人类细胞转铁蛋白受体（ＴＦＲ）的单克隆抗体（ＯＫＴ９）以冰冻切片ＡＢＣ法研究了４１例大肠癌，１９例癌旁组织，２５例正常人大肠膜组织ＴＦＲ的表达。结果表明：癌，癌旁及正常人大肠粘膜组织ＴＦＲ染色阳性率依次为８２．９％，８４．２％和５２％，癌及癌旁组织ＴＦＲ染色阳性率及染色强度均显著高于对照组（Ｐ＜０．０５，Ｐ＜０．００５）。正常人大肠粘膜腺体及形态学正常的癌旁腺体ＴＦＲ染色以细胞基底部及侧浊为