Down-regulation of keratin 14 gene expression after v-Ha-ras transfection of human papillomavirus-immortalized human cervical epithelial cells.

Keratin expression in human cervical squamous cell carcinoma (SCC) lines differed significantly from both normal and human papillomavirus (HPV) immortalized exocervical cells. Keratin 14 (K14) expression, determined by protein synthesis and mRNA levels, was dramatically down-regulated in the cervical SCC lines while keratin 5 (K5) expression was not. K14 expression was similarly down-regulated in an HPV-16 immortalized cervical cell line after tumorigenic transformation with recombinant v-Ha-ras DNA. Cultures derived from nude mouse tumor explants also exhibited an altered keratin profile and the levels of K14 protein synthesis, as well as K14 mRNA, were not detectable. In both cases K5 protein synthesis was not significantly down-regulated. In addition, neoplastic cervical SCC lines exhibited up-regulation of keratins 7, 8, 13, and 19, combined with slight down-regulation of keratins 6 and 16. Epidermal keratinocytes responded in a different manner to exocervical cells. Transfection of human papillomavirus-immortalized epidermal keratinocytes with the BglII N fragment of herpes simplex virus 2 produced a neoplastic cell line, but K5 and K14 expression remained unchanged. Thus, neoplastic transformation of human exocervical cells, both in vivo (spontaneous cervical SCC) and in vitro (HPV-16- and v-Ha-ras-induced cervical SCC), is accompanied by characteristic changes in keratin expression. The specific down-regulation of K14 in these tumorigenic cervical cells, in the absence of significant changes in the expression of K5, implies that the normal coordinate regulation of K5 and K14 gene expression has been uncoupled.     

维甲酸抑制雌激素受体阳性乳腺癌细胞生长机制的研究

目的探讨雌激素受体的表达对维甲酸抑制乳腺癌细胞生长的影响和ＲＡＲα表达的变化。方法雌激素受体阴性的乳腺癌细胞株ＭＤＡ－ＭＢ－２３１细胞,采用分子生物学质粒转染技术,将ＥＲ阴性的乳腺癌细胞ＭＤＡ－ＭＤ－２３１转染成ＥＲ阳性细胞。结果发现ＥＲ转染后的细胞不仅ＲＡＲα的基础表达升高,而且ＲＡ能明显抑制该细胞的生长。同时还发现,无论是已确立的雌激素受体阳性的乳腺癌细胞株,还是雌激素受体转染后的细胞,雌激素都能明显刺激ＲＡＲα的表达。结论雌激素通过ＥＲ上调ＲＡＲα量的表达,在维甲酸对乳腺癌细胞生长的抑制中起着很重要的作用。     

Expression of Fas and anti-Fas-mediated apoptosis in human renal cell carcinoma

Cytokine production in squamous cell carcinoma (SCC) of the head and neck may directly influence the clinical behavior of this aggressive neoplasm since cytokines act as autocrine/paracrine growth factors, as well as immunomodulators. In the present study, the cytokine profiles of 16 SCCs of the head and neck and matched autologous, noncancerous mucosa were determined and compared to normal oral mucosa. Cytokine gene expression in noncancerous mucosa was similar to normal oral mucosa. Levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression were significantly increased in SCC relative to the mean +/- 2 SD of noncancerous mucosa or normal mucosa (p<0.05). Immunohistochemical analyses demonstrated that GM-CSF protein was produced by epithelial cells in the SCC. The overall mRNA expression relative to noncancerous-mucosa or normal mucosa of interleukin-1 beta, interleukin-1 alpha and tumor necrosis factor-alpha was not significantly increased in SCC, however, individual tumors did show selected increases in mRNA expression for these cytokines. These extensive differences in the levels of cytokine mRNA expression could modulate interactions between the tumor cells and the corresponding host cells affecting the behavior of head and neck SCC.     

Transcriptional repression of the human galactocerebrosidase gene in squamous cell carcinomas of the larynx

Alterations of gene expression in squamous cell carcinoma (SCC) cell lines derived from the larynx and keratinocytes derived from adjacent normal mucosa of the larynx have been studied using the mRNA differential display technique. Lane-to-lane comparison of reverse transcribed mRNA showed a strong repression of a 148 bp fragment in SCC cells. The fragment was reamplified and cloned. Sequencing revealed a 99.3% homology with a region in exon 17 of the human galactocerebrosidase (GALC) gene. Northern blot analysis confirmed the differential expression of this gene in both carcinoma cell lines and laryngeal SCC biopsies in contrast with corresponding normal mucosa. To provide further evidence for the differential expression rate, both types of cells were transiently transfected with a 152 bp (-176 to -24) high regulatory promoter element of the 5' flanking region of the GALC gene. Results of 3 independent transfection experiments indicated a 16-fold repression of the GALC gene expression in SCC cells compared with benign keratinocytes. However, neither mutation nor other alterations of the promoter sequence were detected. Expression of the GALC gene is thus greatly affected in SCCs of the larynx.     

Increased nitric oxide levels and iNOS over-expression in oral squamous cell carcinoma

Inducible nitric oxide synthase (iNOS) is responsible for generating high levels of nitric oxide (NO) in tissues. Increased iNOS expression has been demonstrated in a number of carcinomas including head and neck squamous cell carcinoma (SCC). However, iNOS levels have not been evaluated specifically in oral cavity SCC, or in the precancerous lesions that progress to oral SCC. Also, NO levels have not been measured in oral precancerous or cancerous tissues. We therefore measured iNOS mRNA, iNOS protein and NO in oral SCC, oral dysplasias and normal oral epithelium. We used RT-PCR to quantify and compare iNOS mRNA levels in these oral tissue specimens. We found that iNOS mRNA was overexpressed in 41% of oral SCC but in only 8% of dysplasia specimens (P = 0.003). Immunohistochemistry was used to evaluate iNOS protein levels in oral SCC, oral dysplasias and normal oral epithelium. A significantly higher percentage of oral SCC specimens showed the highest level of iNOS staining relative to the oral dysplasias and normal oral epithelial samples (95% of oral SCC, 50% of dysplasias, and only 0% of normal epithelial controls, P < 0.0001). The positive staining for iNOS was limited to the SCC cells. Production of NO from iNOS was quantified using HPLC and found to be significantly higher in oral SCC (1.45 +/- 0.56 microg/ml) than normal epithelial controls (0.43 +/- 0.26 microg/ml) (P = 0.0013). We conclude that iNOS mRNA levels and NO production are significantly increased, in oral SCC compared to oral dysplasias and normal epithelial controls. These findings suggest that increased iNOS expression and the generation of high NO levels might have a role in oral SCC development.     

Expression of E-cadherin, P-cadherin and involucrin by normal and neoplastic keratinocytes in culture

We have compared expression of involucrin, E-cadherin and P-cadherin in cultures of normal keratinocytes and in five different lines derived from squamous cell carcinomas (SCCs), using Northern analysis and immunofluorescence. In normal keratinocytes there was an inverse correlation between P-cadherin and involucrin expression, whereas E-cadherin was expressed by both basal and terminally differentiating cells. In SCC lines involucrin expression was lower than in normal keratinocytes, and there was variable expression of P- and E-cadherin: E-cadherin mRNA levels tended to be lower in SCC lines than in normal keratinocytes, whereas P-cadherin levels were similar. Our results are consistent with observations of cadherin expression in vivo and suggest that the cultures provide a useful experimental model for investigating the role of cadherins in determining the spatial organization of normal and neoplastic keratinocytes.     

Epidermal growth factor receptor gene amplification is correlated with laminin-5 gamma2 chain expression in oral squamous cell carcinoma cell lines.

Both epidermal growth factor receptor (EGFR) gene amplification and laminin (Ln)-5 gamma2 chain overexpression have been reported to be poor prognostic factors in patients with squamous cell carcinoma (SCC) of the head and neck. Here we report our investigation of the relationship between EGFR gene amplification and Ln-5 gamma2 chain expression in seven SCC cell lines, since both epidermal growth factor (EGF) signaling and Ln-5 gamma2 have been reported to be involved in cell motility. The degree of correlation between EGFR gene amplification and Ln-5 gamma2 chain expression was evaluated by Southern and Western blot analyses. EGFR gene amplification was detected in all SCC cell lines at levels 5-50 times those in DNA from normal liver tissue. EGFR gene amplification increased with Ln-5 gamma2 chain protein expression in seven cell lines, showing close correlation between EGFR gene amplification and Ln-5 gamma2 chain protein expression. In order to show the causal relationship, we analyzed the effects of transforming growth factor-alpha (TGF-alpha), tyrosine kinase inhibitor of EGFR, and neutralizing antibody against EGFR, on the expression of Ln-5 gamma2 in these cell lines. In two cell lines in which EGFR gene amplification was low, expression of both protein and mRNA of the Ln-5 gamma2 chain increased in the presence of TGF-alpha, and Ln-5 gamma2 chain expression was inhibited by neutralizing antibody against EGFR. In all cell lines, Ln-5 gamma2 chain expression was inhibited by tyrosine kinase inhibitor which acts selectively on the EGFR signal transduction pathway under the stimulus of TGF-alpha. These results suggest that EGFR gene amplification and the EGFR signaling pathway can act as positive regulators on the induction of the Ln-5 gamma2 chain secreted by tumor cells.     

Gene mutations and increased levels of p53 protein in human squamous cell carcinomas and their cell lines.

Using immunocytochemical and Western blotting techniques we have demonstrated the presence of abnormally high levels of p53 protein in 8/24 (33%) of human squamous cell carcinomas (SCC) and 9/18 (50%) of SCC cell lines. There was a correlation between the immunocytochemical results obtained with eight SCC samples and their corresponding cell lines. Direct sequencing of PCR-amplified, reverse transcribed, p53 mRNA confirmed the expression of point mutations in six of the positive cell lines and detected in-frame deletions in two others. We also detected two stop mutations and three out-of-frame deletions in five lines which did not express elevated levels of p53 protein. Several of the mutations found in SCC of the tongue (3/7) were in a region (codons 144-166) previously identified as being a p53 mutational hot spot in non-small cell lung tumours (Mitsudomi et al., 1992). In 11/13 cases only the mutant alleles were expressed suggesting loss or reduced expression of the wild type alleles in these cases. Six of the mutations were also detected in the SCCs from which the lines were derived, strongly suggesting that the mutations occurred, and were selected, in vivo. The 12th mutation GTG-->GGG (valine-->glycine) at codon 216 was expressed in line SCC-12 clone B along with an apparently normal p53 allele and is to our knowledge a novel mutation. Line BICR-19 also expressed a normal p53 allele in addition to one where exon 10 was deleted. Additionally 15 of the SCC lines (including all of those which did not show elevated p53 protein levels) were screened for the presence of human papillomavirus types 16 and 18 and were found to be negative. These results are discussed in relation to the pathogenesis of SCC and the immortalisation of human keratinocytes in vitro.     

Comparison of integrin expression and terminal differentiation capacity in cell lines derived from oral squamous cell carcinomas

Receptors of the integrin family regulate adhesion and terminaldifferentiation of keratinocytes. In order to investigate thesignificance of changes in integrin expression associated withmalignant transformation we have examined normal human oralkeratinocytes and seven oral squamous cell carcinoma (SCC) lines.Cell surface levels of the     

All-TRANS, 13-CIS and 9-CIS retinoic acids induce a fully reversible growth inhibition in HNSCC cell lines: Implications for in vivo retinoic acid use

Retinoids are a group of vitamin A analogues that have shown promise as chemopreventive and therapeutic agents in many types of malignancy and have been entered in clinical trials with some successful results. To better understand the mechanism that mediates retinoid action and the anti-proliferative effects, we treated 7 human oral squamous-cell carcinoma (SCC) cell lines (FADU, HEp-2, CCL-17, SCC-9, SCC-15, SCC-25 and HN-212) with 10 ⁶ M of all-trans retinoic acid (ATRA), 9-cis and 13-cis retinoic acid (RA) in continuous for different periods of time. We assessed the extent of growth inhibition, the stability of the anti-proliferative effect and the mRNA expression levels (by RT-PCR) of RA receptors (RARs), retinoid X receptors α (RXRα) and cytosolic RA-binding proteins (CRBP I and CRABP II) in treated cells compared with controls. The data obtained showed that all 3 RAs were able to inhibit the cellular growth of the tested cell lines, although to a different extent. The cis compounds were able to inhibit the proliferation of all cell lines, whereas ATRA was ineffective in inhibiting the proliferation of the CCL-17 cell line, which was naturally resistant to ATRA concentrations in the range between 10 ⁵ and 10 ⁶ M. All inhibitory effects were completely reversible since all cell lines restored their normal growth proliferation within few days after drug removal. RT-PCR analysis of the receptor and cell binding protein status of control and treated cells showed a good correlation between growth inhibition and induction of, or increase in, the expression levels of RARβ in RA-treated cells. No differences were observed in RARα and RXRα mRNA expression levels between control and treated cells. CRBP I, CRABP II and RARγ mRNA levels increased in some treated cell lines but not in all. Int. J. Cancer, 70:194–200, 1997. © 1997 Wiley-Liss, Inc.     

Increased ICAM-1 expression in transformed human oral epithelial cells: molecular mechanism and functional role in peripheral blood mononuclear cell adhesion and lymphokine-activated-killer cell cytotoxicity

The intercellular adhesion molecule-1 (ICAM-1, CD54) serves as a counter-receptor for the beta2-integrins, LFA-1 and Mac-1, which are expressed on leukocytes. Although expression of ICAM-1 on tumor cells has a role in tumor progression and development, information on ICAM-1 expression and its role in oral cancer has not been established. Normal human oral keratinocytes (NHOK), human papilloma virus (HPV)-immortalized human oral keratinocyte lines (HOK-16B, HOK-18A, and HOK-18C), and six human oral neoplastic cell lines (HOK-16B-BaP-T1, SCC-4, SCC-9, HEp-2, Tu-177 and 1483) were used to study ICAM-1 expression and its functional role in vitro. Our results demonstrated that NHOK express negligible levels of ICAM-1, whereas immortalized human oral keratinocytes and cancer cells express significantly higher levels of ICAM-1, except for HOK-16B-BaP-T1 and HEp-2. Altered mRNA half-lives did not fully account for the increased accumulation of ICAM-1 mRNA. Adhesion of peripheral blood mononuclear cells (PBMC) to epithelial cells correlated with cell surface ICAM-1 expression levels. This adhesion was inhibited by antibodies specific for either ICAM-1 or LFA-1/Mac-1, suggesting a role for these molecules in adhesion. In contrast, lymphokine-activated-killer (LAK) cell cytotoxic killing of epithelial cells did not correlate with ICAM-1 levels or with adhesion. Nonetheless, within each cell line, blocking of ICAM-1 or LFA-1/Mac-1 reduced LAK cell killing, suggesting that ICAM-1 is involved in mediating this killing.     

口腔黏膜白斑及鳞癌中p57^KIP2蛋白的表达及其意义

目的通过检测p57KIP2蛋白在不同程度异型增生的口腔黏膜白斑及鳞癌中的表达,以探讨其在口腔黏膜白斑及鳞癌中的意义。方法以10例正常口腔黏膜作对照,采用免疫组织化学S-P法回顾性研究79例口腔白斑和67例口腔鳞癌中p57的表达情况。统计分析不同程度异型增生中及鳞癌中p57KIP2蛋白表达的差异。结果 79例口腔黏膜白斑组织中p57KIP2表达率为77.2%（61/79）,显著低于正常口腔黏膜（10/10）（χ2=22.510,P〈0.01）。在白斑组织中,随着异型增生程度增高,p57KIP2蛋白的表达率有下降的趋势（85.0%,70.0%,68.4%）,但差异无统计学意义。p57在轻度异性型增生白斑中的表达显著低于正常黏膜组织（P〈0.05）,而在鳞癌中的表达又显著低于中重度异型增生白斑（P〈0.05）。结论口腔黏膜白斑组织中的p57KIP2蛋白表达显著低于正常口腔黏膜组织,提示从正常口腔黏膜到作为癌前病变的口腔黏膜白斑的发展过程中存在p57KIP2蛋白的表达丧失。p57是预测口腔鳞癌进展和预后的一个生物指标。     

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.