4-hexylresorcinol stimulates the differentiation of SCC-9 cells through the suppression of E2F2, E2F3 and Sp3 expression and the promotion of Sp1 expression

The dormancy-inducing factors of bacteria inhibit tumor cell growth. In the present study, we evaluated the antitumor effects of the dormancy-inducing factor 4-hexylresorcinol (4-HR) using real-time cell electronic sensing (RT-CES) in SCC-9 cells (tongue squamous cell carcinoma cells). Treatment with 4-HR suppressed the growth of SCC-9 cells in a dose-dependent manner. We used a DNA microarray to identify genes that showed a significant change in expression upon 4-HR administration in SCC-9 cells. Among the differentially expressed genes, the protein expression of several cell proliferation related factors, including E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, Sp1 and Sp3, were determined by western blot analyses. Treatment with 4-HR strongly suppressed E2F2 and slightly suppressed E2F3 but did not change the expression of E2F1, E2F4, E2F5 and E2F6 relative to no treatment. Furthermore, 4-HR increased Sp1 expression in a dose-dependent manner and decreased Sp3 expression. Therefore, the ratio of Sp1 to Sp3, an important driving force of epithelial cell differentiation, was drastically increased. Consistent with this observation, 4-HR increased the expression of the epithelial cell differentiation markers involucrin and keratin 10. Together, our results indicate that 4-HR induces the differentiation of SCC-9 via the modulation of the E2F-mediated signaling pathway.     

Expression of keratin proteins in deeply invasive basal and squamous cell carcinoma: an immunohistochemical study

The expression of certain classes of keratin is associated with cell maturation and differentiation. During cell transformation and tumor development, the cell specificity of intermediate filament, keratin, is largely conserved. Taking advantage of this, we utilized monoclonal antikeratin immunohistochemical techniques to examine basal and squamous cell carcinomas as they became deeply invasive. Dyskeratotic keratinocytes and keratin pearls in squamous cell carcinomas (SCCs) stain with antikeratin antisera to larger keratins (65-67 Kd). At the deep tumor margins, SCCs no longer express larger keratins but retain expression of 50, 58 Kd, which are markers of keratinocytes derived from stratified squamous epithelial cells. This selective loss of expression of keratin polypeptide markers of differentiation in SCC is associated with progressively more aggressive biologic behavior as the tumor invades deeper structures such as muscle and bone. Recurrent basal cell carcinoma (BCC) which was of the nodular type when first excised, shows features of morphea-like BCC and of aggressive growth patterns at the deep invasive margin. At these deep margins, some tumors express markers of keratinization (65-67 Kd). While tumor cells retain the specificity of the intermediate filament, keratin, the individual cells express products of differentiation as measured by keratin expression independently of their cytologic atypia. At the deeper invasive margin of the tumor, the neoplastic cells synthesize keratin proteins in an unpredictable fashion.     

E2F suppression and Sp1 overexpression are sufficient to induce the differentiation-specific marker, transglutaminase type 1, in a squamous cell carcinoma cell line

Recently, E2F function has expanded to include the regulation of differentiation in human epidermal keratinocytes (HEKs). We extend these findings to report that in HEKs, Sp1 is a differentiation-specific activator and a downstream target of E2F-mediated suppression of the differentiation-specific marker, transglutaminase type 1 (TG-1). Deletion of elements between -0.084 to -0.034 kb of the TG-1 promoter disabled E2F1-induced suppression of promoter activity. Electrophoretic mobility shift assays (EMSAs) demonstrated that Sp1 and Sp3 bound this region. Protein expression analysis suggested that squamous differentiation was accompanied by increased Sp1/Sp3 ratio. Cotransfection of proliferating HEKs or the squamous cell carcinoma (SCC) cell line, KJD-1/SV40, with an E2F inhibitor (E2Fd/n) and Sp1 expression plasmid was sufficient to activate the TG-1 promoter. The suppression of Sp1 activity by E2F in differentiated cells appeared to be indirect since we found no evidence of an Sp1/E2F coassociation on the TG-1 promoter fragment. Moreover, E2F inhibition in the presence of a differentiation stimulus induced Sp1 protein. These data demonstrate that (i) Sp1 can act as a differentiation stimulus, (ii) E2F-mediated suppression of differentiation-specific markers is indirect via Sp1 inhibition and (iii) a combination of E2F inhibition and Sp1 activation could form the basis of a differentiation therapy for SCCs.     

Cell cycle genes as targets of retinoid induced ovarian tumor cell growth suppression.

We have examined the effect of all-trans-retinoic acid (RA) on cell cycle gene expression in RA sensitive CA-OV3 and RA resistant SK-OV3 ovarian carcinoma cell lines. Gene expression was analysed by multiprobe RNAse protection, Western blotting and in vitro kinase assays. No differences were observed between RA sensitive and RA resistant ovarian carcinoma cells in the levels of expression of many cell cycle genes including cyclin A, B and E, cdk 2,4 and 6, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, DP-1 and DP-2. However, RA sensitive CA-OV3 cells expressed higher levels of p53, p27, p21, and p16 compared to RA resistant SK-OV3 cells. In addition, RA treatment of CA-OV3 cells resulted in a significant decrease in hyperphosphorylated RB and RB-2/p130 and corresponding significant increases in the levels of hypophosphorylated and/or partially phosphorylated RB-2/p130 protein and hypophosphorylated RB. Also, RA treatment increased expression of the cdk inhibitor p27 and decreased activity of cdk 2, cdk 4 and cdk 6. Finally, amounts of p27-cyclin E and RB-2/p130-E2F4 complexes were found to increase in CA-OV3 cells growth arrested by RA. These results suggest that the pocket protein pathways are critical targets for retinoid suppression of ovarian carcinoma cell growth.     

Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection.

Human papillomavirus (HPV) DNAs are detected in most genital dysplasias and cancers, suggesting that these viruses perturb epithelial growth and differentiation. The E6 and E7 genes of HPV type 18 induce immortality in keratinocytes cultured from genital tract epithelia, and the immortal cell lines display aberrant squamous differentiation. To examine whether the E6 and E7 proteins directly alter keratinocyte growth and differentiation, high-titer recombinant retroviruses were constructed for efficient transfer and expression of HPV-18 genes E6, E6* and E7 in cultures of normal human keratinocytes. Infection with retroviruses encoding E6 and E7 stimulated cell proliferation, reduced the requirement for bovine pituitary extract and induced immortality. E6 and E7 also delayed but did not prevent the onset of terminal squamous differentiation. The magnitude of effects on growth and differentiation of cultured cells was directly related to levels of E7 protein expression. Thus, expression of the HPV-18 E6 and E7 genes stimulates cell proliferation and delays differentiation of keratinocytes in vitro.     

E2F-1和Rb基因在食管鳞状细胞癌中的 表达及临床意义

 目的 研究E2F-1和Rb基因在食管鳞状细胞癌中的表达情况,探讨其在食管鳞状细胞癌发生发展中的规律与临床病理关系。方法 应用免疫组化方法检测60例食管鳞状细胞癌和50例正常食管组织中E2F-1和Rb基因的表达情况。结果 食管鳞状细胞癌中E2F-1的阳性表达率为73.3％,显著高于癌旁正常组织黏膜中的36.0％（P<0.01）,E2F-1的表达与食管鳞状细胞癌的分期、分化及淋巴结转移有相关性（P<0.05）。Rb在食管鳞状细胞癌中的阴性表达率为30.0％,显著高于癌旁正常组织中的12.0％（P<0.05）,Rb在食管鳞状细胞癌中的表达与分化及淋巴结转移有关（P<0.05）,但尚不能认为与分期有关。结论 E2F-1和Rb在食管鳞状细胞癌中的表达呈负相关,即E2F-1过度表达而Rb表达有一定程度缺失,在正常食管组织中的表达亦有差异。     

Differential expression of apoptosis, cell cycle regulation and intermediate filament genes in oral squamous cell carcinomas associated with toombak use in Sudan

Previously we used microarray genomic hybridization technology to explore genome-wide profiles of chromosomal aberrations in samples of oral squamous cell carcinomas (OSCCs) and paired normal controls. Based on these findings, 9 genes related to apoptosis, cell cycle regulation and intermediate filament proteins were selected and their differential expression status was examined by real-time quantitative RT-PCR in 26 samples of Sudanese OSCCs and their matched normal controls. The findings were correlated with the habit of toombak use. The mRNA levels of Bcl2, keratin 1, keratin 13 and p53 were significantly lower and the level of survivin was significantly higher in the OSCC samples of the toombak users compared to their paired control samples. A significant down-regulation in keratin 1 and keratin 13 expression levels was found in the OSCC samples of the nontoombak users compared to their normal control samples. The differential expression of genes related to apoptosis, cell cycle regulation and types I and II keratin could be useful diagnostic markers and provide valuable information for the understanding of oral malignancy in relation to toombak use.     

Rac1 regulates skin tumors by regulation of keratin 17 through recruitment and interaction with CD11b+Gr1+ cells

Rac1 is a member of the Rho family of small GTPases that control cells proliferation, differentiation, migration, and inflammation. Rac1 is crucial in tumorigenesis and development. Keratin17 and CD11b+Gr1+ cells are considered to regulate skin inflmmation. Here we discuss the regulation of Rac1 on skin tumor formation and its relationship. In samples from human skin squamous cell carcinoma (SCC), Rac1 activity was higher in cancer tissues than in normal skin and activity correlated with keratin 17 overexpression. In a DMBA/TPA-induced mouse skin tumor model, inhibition of Rac1 activity and depletion of CD11b+Gr1+ cells resulted in significant tumor formation. TPA induced recruitment of CD11b+Gr1+ cells into dermis; however, Rac1 inhibitor abolished this recruitment. In vitro, Rac1 induced interferon (IFN) and interlukin (IL6) production in keratinocytes, repression of keratin 17 inhibited IFN and IL6 production induced by Rac1. Moreover, both inhibition of Rac1 activity and repression of keratin 17 restricted proliferation and induction of differentiation in keratinocytes. Coculture of CD11b+Gr1+ cells with keratinocytes activated Wnt pathway in keratinocytes, resulting in enhanced Rac1 activity, overexpression of keratin 17, and hyperproliferation of keratinocytes. Our results suggested that hyperactive Rac1 recruited and interacted with CD11b+Gr1+ cells, inducing keratin 17-regulated inflammation and promoting skin tumor formation.     

Keratin and involucrin immunohistochemistry of nasopharyngeal carcinoma

Forty nasopharyngeal carcinomas (NPC) were studied by immunohistochemistry using an antibody to involucrin and the following three keratin antibodies: (1) an antibody to low molecular weight keratin reactive with nonsquamous epithelium, (2) a high molecular weight keratin antibody reactive with suprabasal squamous epithelium, and (3) a keratin antibody reactive with full thickness stratified epithelium. In its pattern of reactivity, the last antibody overlaps the low and high molecular weight keratin antibodies and is used as a broad spectrum keratin antibody. By World Health Organization (WHO) classification, the cases in this article included eight keratinizing squamous cell carcinomas, eight nonkeratinizing carcinomas, 20 undifferentiated carcinomas, and four adenocarcinomas. The antibody to broad spectrum keratin had an overall sensitivity of 87.5% and was positive in all eight keratinizing squamous cell carcinomas, seven nonkeratinizing carcinomas (87.5%), 18 undifferentiated carcinomas (90%), and two adenocarcinomas (50%). Low molecular weight keratin antibody stained one additional NPC, which was negative when broad spectrum keratin antibody was used. Involucrin and high molecular weight keratin antibodies demonstrated near parallel staining in all histologic classes; there was marked localization to areas of squamous differentiation. While involucrin is a marker for foci of greater squamous differentiation, broad spectrum keratin antibody may aid in the diagnosis of all histologic subtypes of NPC.     

E2F3在喉鳞状细胞癌中的表达及临床意义

目的：探讨喉鳞癌中E2F3的表达与临床意义,为喉鳞癌的诊断、评估及治疗寻找辅助生物学指标。方法：采用免疫组化方法检测喉鳞癌组织及癌旁正常切缘组织中E2F3表达,结合喉鳞癌各临床参数,用SPSS16.0软件包进行统计分析E2F3表达情况与喉鳞癌各临床特征之间的关系。结果：E2F3在喉鳞癌与癌旁正常切缘中的阳性表达率分别为90.44%（123/136）和27.85%（22/79）,差异具有显著统计学意义（P〈0.001）;E2F3高表达与喉鳞癌患者年龄、临床分期及分型均相关（P〈0.05）。结论：E2F3核表达可能参与喉鳞癌发生发展过程;E2F3浆表达可能贯穿于喉鳞癌发生的整体过程。     

Keratin expression in normal uterine cervical epithelium and carcinomas of cervical origin

The immunohistochemical expression of keratins 7, 8, 10, 13, 14, 17, 18 and 19 was examined in formalin-fixed paraffin-embedded tissues of normal uterine cervical epithelium and carcinomas of cervical origin (4 squamous cell carcinoma in situ, 17 squamous cell carcinoma, 9 adenocarcinoma, and 1 adenoid basal carcinoma). A panel of 8 monoclonal antibodies capable of recognizing 8 individual keratin subtypes was employed using microwave oven heating and a labeled streptavidin biotin method. Ectocervical squamous epithelium expressed keratins 14 and 19 in the basal cell layer, and keratins 10 and 13 in the suprabasal cell layer. Endocervical columnar cells were found to express keratins 7, 8, 18 and 19, whereas the reserve cells expressed keratins 7, 8, 14, 17, 18 and 19. Most of the squamous cell carcinomas, both keratinizing and non-keratinizing, as well as the carcinoma in situ revealed a keratin phenotype detected in normal ectocervical squamous cells (keratins 10, 13, 14 and 19) and endocervical subcolumnar reserve cells (keratins 7, 17 and 18). The adenocarcinomas, both endocervical and endometrial type, were positive for keratins 7, 8, 17, 18 and 19. The adenoid basal carcinoma expressed all the keratins examined including the expression of reserve cell keratin. Reserve cell keratins were found mostly in squamous cell carcinomas, adenocarcinomas and adenoid basal carcinoma of cervical origin. Therefore, the keratin expression pattern indicates the origin of a variety of carcinomas of the uterine cervix from a common progenitor, endocervical reserve cells.