Telomerase activity, estrogen receptors (α, β), Bcl-2 expression in human breast cancer and treatment response

BACKGROUND: The mechanism for maintaining telomere integrity is controlled by telomerase, a ribonucleoprotein enzyme that specifically restores telomere sequences, lost during replication by means of an intrinsic RNA component as a template for polymerization. Among the telomerase subunits, hTERT (human telomerase reverse transcriptase) is expressed concomitantly with the activation of telomerase. The role of estrogens and their receptors in the transcriptional regulation of hTERT has been demonstrated. The current study determines the possible association between telomerase activity, the expression of both molecular forms of estrogen receptor (ERα and ERβ) and the protein bcl-2, and their relative associations with clinical parameters. METHODS: Tissue samples from 44 patients with breast cancer were used to assess telomerase activity using the TRAP method and the expression of ERα, ERβ and bcl-2 by means of immunocytochemical techniques. RESULTS: Telomerase activity was detected in 59% of the 44 breast tumors examined. Telomerase activity ranged from 0 to 49.93 units of total product generated (TPG). A correlation was found between telomerase activity and differentiation grade (p = 0.03). The only significant independent marker of response to treatment was clinical stage. We found differences between the frequency of expression of ERα (88%) and ERβ (36%) (p = 0.007); bcl-2 was expressed in 79.5% of invasive breast carcinomas. We also found a significant correlation between low levels of telomerase activity and a lack of ERβ expression (p = 0.03). CONCLUSION: Lower telomerase activity was found among tumors that did not express estrogen receptor beta. This is the first published study demonstrating that the absence of expression of ERβ is associated with low levels of telomerase activity

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression

Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene

The original publication can be found at www.springerlink.comThe periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10–20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 μg/ml ascorbic acid and 2 mM β-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.Shinsuke Fujii, Hidefumi Maeda, Naohisa Wada, Yoshio Kano and Akifumi Akamin