Vitamin D receptor expression in normal, premalignant, and malignant human lung tissue.

BACKGROUND: There is a strong interest in identifying chemopreventive agents that might help decrease the burden of lung cancer. The active metabolite of vitamin D, 1,25-dihydroxycholecalciferol (calcitriol), has been shown to have antiproliferative effects in several tumor types, mediated by the vitamin D receptor (VDR). This is the first comprehensive survey of VDR expression in a series of human lung tissues, including normal and premalignant central airway biopsies and lung tumors. METHODS: Immunohistochemical expression of nuclear and cytoplasmic VDR was examined in 180 premalignant or malignant bronchial biopsies from bronchoscopy of 78 high-risk individuals at the Roswell Park Cancer Institute and also in 63 tumor samples from 35 lung cancer patients from the University of Chicago Hospitals. Associations between clinicopathologic data and VDR expression were examined. RESULTS: VDR expression was present in many samples. In biopsies, VDR was commonly detected throughout the full epithelial layer. Most histologically normal (60%, 53 of 88) and metaplastic (61%, 39 of 64) samples had moderate to high nuclear intensity; dysplastic samples mostly had low nuclear intensity (10 of 18, 55%). In tumor samples, 62% (38 of 61) were lacking cytoplasmic VDR, with nuclear expression present in 79%(49 of 62). Analysis of all samples revealed a positive linear trend between proportion of samples with greater nuclear than cytoplasmic intensity and increasing histologic grade (P < 0.01). CONCLUSIONS: VDR expression spanned the lung carcinogenesis spectrum. Nuclear expression was similar across various histologies, whereas cytoplasmic expression decreased with increasing histologic grade. These results indicate that there is potential for the use of calcitriol as a chemopreventive agent against the development of lung cancer.     

Comparison of T-antigen expression in normal, premalignant, and malignant human colonic tissue using lectin and antibody immunohistochemistry

The Thomsen-Friedenreich antigen has been implicated as a cancer-associated antigen in some human organs including the colon. Most previous studies of Thomsen-Friedenreich antigen expression in the colon used peanut agglutinin (PNA) to identify the immunodeterminant in tissues. However, evidence from other organs suggests that anti-T antibodies have specificities which differ from those of peanut lectin. To elucidate the nature of the T-immunodeterminant in colonic mucosa, we compared staining by PNA to that of a polyclonal (PAb) and monoclonal (MAb) anti-T antibody. PNA demonstrated the best sensitivity (91%) in cancer tissues but the lowest specificity (68%) in normal mucosa. Staining with MAb was only 76% sensitive but 100% specific. Sensitivity and specificity of PAb were intermediate between PNA and MAb. MAb stained fewer adenomatous polyps than either PNA or PAb, but staining appeared to correlate with premalignant features of the polyps. PNA-binding sites were more prevalent than either PAb or MAb in hyperplastic polyps. Cell cytoplasm was stained by both antibodies more often than by PNA. The majority of fetal colonic specimens stained with all three reagents suggesting that Thomsen-Friedenreich antigen may be an oncodevelopmental antigen in human colon. Differences in staining patterns in some tissues may be due to different antigenic specificities among PNA, PAb, and MAb.     

Differential gene expression of Wnt signaling pathway in benign, premalignant, and malignant human breast epithelial cells

To gain insight into the role of gene expression alterations in breast cancer progression, we conducted a comprehensive gene expression analysis of a series of cell lines derived from MCF10A, which include benign MCF10A cells, premalignant AT, and malignant CA1a tumor cells. We analyzed gene expression variation using the Agilent Human Genome Oligo Microarray with the goal of identifying gene-specific expression change events. In addition to a previously noted overexpression in oncogene MDM2, HRAS, and PCNA, our studies identified overexpression of Wnt signaling pathway in malignant breast cell lines. The Kaplan?CMeier plot showed that high c-Myc expression in breast cancer was associated with tumor progression and the patient??s poor survival. This study showed that the Wnt pathway has further provided a basis for the development of potential biomarker for breast cancer prognosis.     

hMLH1蛋白在食管鳞状细胞癌和不典型增生组织中的表达

目的：探讨同一食管癌患者食管的鳞状细胞癌组织、不典型增生组织、正常鳞状上皮组织中错配修复基因1（human mutl homolog 1，hMLH1）的蛋白表达情况，深入研究它与食管鳞状细胞癌发生发展的关系。方法：取92例食管鳞状细胞癌患者的病变组织，其病理切片中鳞状细胞癌组织、不典型增生组织和正常鳞状上皮组织均存在，采用免疫组织化学的方法，检测hMLH1蛋白在3种不同组织中的表达，并分析其与性别、年龄、分化程度、浸润深度、肿瘤分期、淋巴结转移等临床病理参数之间的关系。结果：hMLH1蛋白在食管鳞状细胞癌组织、不典型增生组织、正常鳞状上皮组织细胞核中的阳性表达率分别为36．96％、56．52％、84．78％，鳞状细胞癌组织和不典型增生组织均显著低于正常食管鳞状上皮组织（P〈0．01）。鳞状细胞癌组织中hMLH1蛋白表达阴性者，年龄较阳性者大（P〈0．05）；hMLH1蛋白表达与肿瘤分期、淋巴结转移等有明显相关性（P〈0．05）。结论：hMLH1基因缺失可能在早期就参与了食管鳞状细胞癌的发生过程，hMLH1蛋白可能抑制或延缓食管鳞状细胞癌的发生和发展。     

Constitutive expression of human keratin 14 gene in mouse lung induces premalignant lesions and squamous differentiation

Squamous cell carcinoma accounts for 20% of all human lung cancers and is strongly linked to cigarette smoking. It develops through premalignant changes that are characterized by high levels of keratin 14 (K14) expression in the airway epithelium and evolve through basal cell hyperplasia, squamous metaplasia and dysplasia to carcinoma in situ and invasive carcinoma. In order to explore the impact of K14 in the pulmonary epithelium that normally lacks both squamous differentiation and K14 expression, human keratin 14 gene hK14 was constitutively expressed in mouse airway progenitor cells using a mouse Clara cell specific 10 kDa protein (CC10) promoter. While the lungs of CC10-hK14 transgenic mice developed normally, we detected increased expression of K14 and the molecular markers of squamous differentiation program such as involucrin, loricrin, small proline-rich protein 1A, transglutaminase 1 and cholesterol sulfotransferase 2B1. In contrast, wild-type lungs were negative. Aging CC10-hK14 mice revealed multifocal airway cell hyperplasia, occasional squamous metaplasia and their lung tumors displayed evidence for multidirectional differentiation. We conclude that constitutive expression of hK14 initiates squamous differentiation program in the mouse lung, but fails to promote squamous maturation. Our study provides a novel model for assessing the mechanisms of premalignant lesions in vivo by modifying differentiation and proliferation of airway progenitor cells.     

Mucin gene expression in colonic tissues and cell lines.

Complementary DNA clones encoding four different mucin core peptides have been isolated. However, the expression of these mucin genes in the colon has not been systematically studied. The present investigation used Northern blot analysis to study the expression of MUC1, MUC2, MUC3, and MUC4 mRNA in paired normal and cancerous colonic tissues, and nine colon cancer cell lines. Results were correlated with the clinicopathological features of the tumors and with the immunohistochemical expression of several carbohydrate tumor-associated antigens that may reside on mucins. MUC1 mRNA was expressed in all colonic tissues, and levels in paired normal and cancer tissues were similar in most cases. MUC2 and MUC3 mRNAs were expressed in both normal and cancer tissues, but levels were often decreased in the cancers. MUC4 mRNA was present in normal mucosa with comparable or sometimes greater expression in cancers. There was no apparent correlation between the expression of any particular mucin gene or pattern of mucin genes and the site, stage, or histological type of tumor. In addition, the expression of mucin-associated carbohydrate antigens did not correlate with any individual mucin gene or group of mucin genes. In colon cancer cell lines all four MUC genes were expressed rather weakly or not at all. These results indicate that the human colon expresses a broad repertoire of mucin genes which are differentially regulated in malignancy. Whether this differential regulation of mucin genes affect the behavior of the tumor and results in the altered glycosylation commonly seen in these requires further investigation.     

Bisphosphonates directly regulate cell proliferation, differentiation, and gene expression in human osteoblasts

Bisphosphonates are widely used clinically to treat bone diseases in which bone resorption is in excess. However, the mechanism of bisphosphonate action on bone is not fully understood. Studies of direct action of bisphosphonates on bone have been limited mainly to their effects on bone-resorbing osteoclast cells, with implications that some activity may be mediated indirectly through paracrine factors produced by the bone-forming osteoblast cells. Little is known about the direct effects of bisphosphonates on osteoblasts. In this report, the direct actions of several bisphosphonates on cell proliferation, gene expression, and bone formation by cultured human fetal osteoblasts were examined. Osteoblast cell proliferation was decreased, and cytodifferentiation was increased in a dose-dependent manner in cultures treated with the bisphosphonate pamidronate. In addition, pamidronate treatment increased total cellular protein, alkaline phosphatase activity, and type I collagen secretion in osteoblasts. Consistent with the above-mentioned findings, the rate of bone formation was also increased in osteoblasts cultured with pamidronate. The actions of two other bisphosphonates, the weak-acting etidronate and the potent new analogue zoledronate, were also compared with the action of pamidronate on proliferation of immortalized human fetal osteoblast (hFOB) cells and rate of bone formation. Pamidronate and zoledronate decreased hFOB cell proliferation with equal potency, whereas etidronate decreased proliferation only at much higher concentrations. Studies comparing EDTA and etidronate indicate that etidronate may act indirectly on the hFOB cells by reducing free divalent ion concentrations, whereas pamidronate and zoledronate appear to act on the hFOB cells by a direct action. Both pamidronate and zoledronate increase hFOB cell bone formation, whereas no increase is observed with etidronate and EDTA. Taken together, these observations strongly suggest that treatment with pamidronate or zoledronate enhances the differentiation and bone-forming activities of osteoblasts.     

Modulation of Ki67, p53 and RARbeta expression in normal, premalignant and malignant human oral epithelial cells by chemopreventive agents

BACKGROUND: Aberrant expression of Ki67, p53 and RARbeta are characteristic of many tumor types including those of the oral cavity. Chemopreventive agents may act by modulating their expression to more normal levels. MATERIALS AND METHODS: The effects of 21 chemopreventive agents on the expression of Ki67, p53 and RARbeta were determined using a human in vitro model of normal, premalignant and malignant oral epithelial cell lines. RESULTS: Ki67 and mutant p53 (mtp53) were overexpressed in both the premalignant and malignant cell lines, whereas expression of RARbeta was high in the normal, low in the premalignant and not detectable in the malignant cell lines. Most of the agents selectively inhibited the expression of Ki67 in the premalignant and malignant cell lines. Eight of the 21 agents increased, while four agents decreased, the levels of mtp53 protein in the premalignant cell line. In the malignant cell line, five of the agents increased, while ten agents decreased mtp53 protein levels. The agents increased RARbeta expression to near normal levels in the premalignant cell line. CONCLUSION: The data suggest that the suppression of Ki67 and mtp53 are good indicators of the effectiveness of agents in premalignant and malignant oral cells, whereas the enhancement of RARbeta is a measure of effectiveness in premalignant oral cells.     

Mucin expression profile in Barrett's, dysplasia, adenocarcinoma sequence in the esophagus

BACKGROUND: The molecular events that accompany the progression to adenocarcinoma (ADC) of the esophagus are poorly understood. Aberrant mucin receptor expression can contribute to increased cell growth and metastatic ability. AIM: The aim of this study was to establish a pattern for mucin (MUC) gene expression in the esophageal mucosa under normal and pathological conditions. SETTING: University Hospital Cancer Center Laboratory. Archived tissue samples studied in a retrospective fashion. MATERIALS AND METHODS: Tissue samples were obtained from the archives of patients with histological evidence of Barrett's esophagus (BE) progressing to ADC. Immunohistochemical analysis was performed using mouse monoclonal antibodies for MUC1, MUC2, MUC5AC, MUC6. Semiquantitative scoring of histological staining was performed using a linear scoring system: 0-staining absent; 1-staining in 0-25%; 2-staining in 25-50%; and 3-staining in 50-75% of the epithelium. The Binomial test was used to explore trends and differences in frequency of mucin expression along the pathological sequence. RESULTS: Only mild superficial staining of MUC1 was seen in normal squamous epithelium. MUC1 and MUC2 were uniformly expressed in all samples (7/7) of BE and dysplasia (P=0.008). MUC1 expression was upregulated (7/7) in progression to adenocarcinoma (P=0.008). The secretory mucins, MUC5AC and MUC6 showed a decrease in expression with progression from BE to dysplasia to ADC (P< 0.05). CONCLUSIONS: Downregulation of MUC5AC and MUC6 decreases mucosal protection against gastric acid. Increasing MUC1 expression is associated with progression from dysplasia to ADC. Upregulation of MUC2 reflects intestinal metaplasia in BE.     

Expression of cell cycle control proteins in normal epithelium, premalignant and malignant lesions of oral cavity

In this study, we examined the expression of cyclins, cyclin dependent kinase (CDKs) and CDK inhibitors by immunohistochemical analysis in 20 normal mucosa, 42 epithelial dysplasia (ED), and 117 oral squamous cell carcinoma. Neither Cyclin D1 nor CDK2 were detectable in normal tissue and ED. Their presence, however, was detectable in squamous cell carcinoma (SCCs) (Cyclin D1, 35.9%; CDK2, 66.7%). Cyclin E was detectable in 57.1% of severe ED and 62.8% of SCCs. For the CDK inhibitors, these proteins were detectable in all normal mucosa and most of the mild and moderate ED. For severe ED, expression of these proteins was not observed in some cases (p12(DOC-1), 14.3%; p16(INK4A), 28.6%; p27(KIP1), 7.1%). For SCCs, the expression of p12(DOC-1) was lost in 71.8%, p16(INK4A) in 69.2% and p27(KIP1) in 35.9%. These results suggest that elevated expression of cyclin D1, cyclin E, CDK2 and loss of p12(DOC-1), p16(INK4A) and p27(KIP1) may contribute to the multistep nature of oral carcinogenesis.     

Differential responses of normal, premalignant, and malignant human bronchial epithelial cells to receptor-selective retinoids.

Using an in vitro lung carcinogenesis model consisting of normal, premalignant, and malignant human bronchial epithelial (HBE) cells, we analyzed the growth inhibitory effects of 26 novel synthetic retinoic acid receptor (RAR)- and retinoid X receptor (RXR)-selective retinoids. RAR-selective retinoids such as CD271, CD437, CD2325, and SR11364 showed potent activity in inhibiting the growth of either normal or premalignant and malignant HBE cells (IC50s mostly <1 microM) and were much more potent than RXR-selective retinoids. Nonetheless, the combination of RAR- and RXR-selective retinoids exhibited additive effects in HBE cells. As the HBE cells became progressively more malignant, they exhibited decreased or lost sensitivity to many retinoids. The activity of the RAR-selective retinoids, with the exception of the most potent retinoid, CD437, could be suppressed by an RAR panantagonist. These results suggest that: (a) RAR/RXR heterodimers play an important role in mediating the growth inhibitory effects of most retinoids in HBE cells; (b) CD437 may act through an RAR-independent pathway; (c) some of the RAR-selective retinoids may have the potential to be used in the clinic as chemopreventive and chemotherapeutic agents for lung cancer; and (d) early stages of lung carcinogenesis may be responsive targets for chemoprevention by retinoids, as opposed to later stages.     

ckshs expression is linked to cell proliferation in normal and malignant human lymphoid cells

Cyclin kinase sub‐units (CKS) are known to interact with cyclin‐dependent kinases (CDKs), but their functions are not completely understood and their expression in human tissues is not documented. For analyzing relationships of CKS with cell proliferation and/or with differentiation, we investigated the expression of ckshs1 and ckshs2 in normal and malignant human lymphoid cells. ckshs1 and ckshs2 expression appeared to be related to cell proliferation: (i) mRNAs increased with stimulation of normal peripheral‐blood lymphocytes, and from the G₁ to the SG₂M phase in elutriated cells; (ii) P9 proteins were also induced by lymphocyte stimulation and were localized in nucleus where phosphorylated forms of CDK1 were also found; (iii) in vitro, the phosphorylated forms of CDK1 and CDK2 were preferentially linked to CKS. Among 45 patients presenting acute or chronic lymphoid malignancy, ckshs1 and ckshs2 mRNAs varied in a similar way and were significantly correlated to cell proliferation (p < 0.0001). When analysis was restricted solely to acute lymphoblastic leukemia (ALL) this correlation was still found and ckshs1 and ckshs2 were significantly more expressed in T‐cell ALL than in B‐cell‐lineage ALL. These results confirm relationships between ckshs expression and cell proliferation, and pose the question of a link with cell differentiation. Int. J. Cancer 82:98–104, 1999. © 1999 Wiley‐Liss, Inc.     

Differential response of normal, premalignant and malignant human oral epithelial cells to growth inhibition by chemopreventive agents

Squamous cell carcinoma (SCC) of the oral cavity is a multistep process, progressing through a series of discrete, irreversible and complementary alterations in genes that control cell growth, death, and differentiation. In the premalignant state, the oral mucosa progresses through various grades of epithelial dysplasia, with the potential to convert to SCC. Chemopreventive strategies are designed to suppress, reverse, or prevent the formation of premalignant lesions and their subsequent progression to SCC. In the present study, we determined the growth inhibitory effect of 21 chemopreventive agents in a cell culture model using normal, premalignant, and malignant human oral mucosal cell lines. There were significant differences in the growth inhibitory responses of these cell lines to selected retinoids and non-retinoid analogs. Among the retinoids tested, the synthetic retinamides, as a class, showed selective growth inhibition of both premalignant and malignant cells compared to normal human oral epithelial cells in culture. Within the retinamide class, 2CPR exhibited the greatest selectivity in the growth inhibition of premalignant and malignant cells. Among the non-retinoids analyzed, DFMO was a moderate to potent inhibitor of malignant and premalignant oral cell growth, respectively, and stimulated normal oral cell growth at low concentrations. Using this in vitro approach, we have identified several potential chemopreventive agents for oral cancer as selective growth inhibitors of premalignant ahd malignant human oral mucosa cells.     

Differential expression of c-myc gene and c-fos gene in premalignant and malignant tissues from patients with familial polyposis coli

The expression of 8 oncogenes and the structures of 19 oncogenes were analyzed in 15 adenocarcinomas (12 primary and 3 metastatic), 18 adenomatous polyps, and 18 normal colonic mucosae derived from 19 patients with familial polyposis coli. The expression of c-myc gene was most elevated in carcinoma, and moderately elevated in adenoma, compared with corresponding normal colonic mucosa. In contrast, the expression of c-fos gene was markedly decreased in all samples of adenoma and carcinoma, compared with that of normal colonic mucosa. These characteristic expression patterns of c-myc and c-fos genes were revealed not only in familial polyposis coli but also in cases of nonhereditary colon carcinoma. Structures of the 19 oncogenes were not modified in either adenoma or carcinoma, except for amplification of the c-myc gene detected in one carcinoma, but not in adenoma, from the same patient. Analyses of the amplified c-myc gene suggest that gene duplication may relate to the mechanism of gene amplification. Thus, the enhanced expression of c-myc gene in adenoma and carcinoma may reflect the proliferative activity, while the c-fos gene may be a prerequisite to stabilize the state of terminal differentiation of colonic epithelial cells.     

Cloning of 67-kDa laminin receptor cDNA and gene expression in normal and malignant cell lines of the human lung.

Cell-adhesive protein laminin and its specific receptor play an important role in the processes of cancer proliferation, invasion and metastasis. In the present study, we cloned the cDNAs of the 67-kDa laminin receptor both from a human lung cell line (IMR90) and from a human lung cancer cell line (SBC3), and determined the nucleotide sequences. In comparison with both cDNA sequences of the protein-coding region, three nucleotide differences were found. These differences in the secondary structure of the protein, however, were caused by nucleotide substitutions. It was also demonstrated that the level of 67-kDa-laminin receptor mRNA was higher in SBC3 than in IMR90.     

Immunohistochemical localization of epidermal growth-factor and its receptor in normal, premalignant and malignant oral-mucosa

An important growth factor involved in epithelial carcinogenesis is the epidermal growth factor (EGF). The present study analyze the expression pattern of EGF and its receptor (EGFR) in different stages of tumour progression in oral mucosa (normal epithelium, non-dysplastic and dysplastic leukoplakias and carcinomas). Alterations in expression pattern of EGFR was not significant in the various tissues from normal mucosa to malignancy. In all these stages EGFR positivity was confined to the immature basal or basaloid cells. EGF however showed marked alterations in expression in different stages of tumour progression in oral mucosa. Most of the malignant cells were positive for EGF antibodies while, only a few lower layers of cells were stained in normal mucosa and in dysplastic leukoplakia lesions, more number of cells expressed EGF than normal tissue. The present study thus shows the autocrine role of the EGF and EGFR and the possibility of using EGF as a marker for tumour progression in oral mucosa.