A study of Ki-67, c-erbB2 and cyclin D-1 expression in CIN-I, CIN-III and squamous cell carcinoma of the cervix

The histological criteria for cervical intraepithelial neoplastic lesions and their follow-ups have been established, but their reproducibility, specificity and sensibility are not certain. Immunohistochemical markers provide more information on each specific case, in order to facilitate its classification and, eventually, its prognosis. Using immunohistochemical techniques, this study analyzes the prognostic value of three markers (Ki-67, c-erbB2 and Cyclin D1) in cases of low grade squamous intraepithelial neoplasia (CIN-I), high grade squamous intraepithelial neoplasia (CIN-III), and infiltrating squamous cell carcinoma (SCC) taken from a group of cervical samples. In situ hybridization was performed in order to detect high-risk HPV. High risk HPV was demonstrated in 82%, 89% and 100% of the LGSIL, HGSIL and SCC cases, respectively. C-erbB2 expression was detected in 9%, 33% and 50% of the LSIL, HGSIL and SCC cases, respectively. The Ki-67 LI was 25%, 68% and 65.5% in the LGSIL, HGSIL and SCC cases, respectively. Nuclear Cyclin D1 expression was seen in 82%, 11% and 30% of the CIN-I,CIN-III and SCC cases, respectively. We observed that the cytoplasmic cyclin D1 expression increased with the severity of the lesion instead of the nuclear expression decreasing with the progression of the pathology. Nuclear and cytoplasmic Cyclin D1 expression seemed to be related to HPV high risk infection. We concluded that Cyclin D1, cerbB2 and The Ki-67 LI expression changed in relation to the severity of the lesion and that they could be helpful in making a differential diagnosis.     

Cervical intraepithelial neoplasia: Telomerase activity and splice pattern of hTERT mRNA

Cervical cancers are characterized by the persistence of human papilloma virus (HPV) genome that is found in tissue samples starting from the early stages of tumor progression. Just like in other tumors, the activation of telomerase was observed in cervical carcinomas, but information about its expression was controversial. The aim of this study is to find possible correlations between the presence of HPV sequences, activity of telomerase and expression of different spliced forms of hTERT RNA in cervical intraepithelial neoplasias (CIN). The results show that HPV DNA is present in 60% of normal tissue adjacent to CIN lesions and up to 84% in CIN samples. Telomerase activity was found in 28% of adjacent normal tissue and in 68% of CIN II–III. hTERT RNA that encodes an active enzyme was present almost in all CIN samples. Variations in levels of telomerase activity are possibly not regulated by the splicing forms of hTERT mRNA with deletions.     

Telomerase activity in cervical smears.

It is well known that almost all carcinoma cells including those of the uterine cervix have re-established their telomerase activity. However, until now there is no conclusive picture on the telomerase activity in cervical dysplasias and about their relationship to HPV infection. To investigate this question, material from 34 patients (15 with normal epithelium, 11 with LGSIL, 8 with HGSIL) obtained by conventional cervical brushing was used and subjected to non-radioactive TRAP-ELISA (Boehringer Mannheim). The HPV analysis was performed by PCR on formalin-fixed, paraffin-embedded biopsy material obtained after cytological investigation. We could show that telomerase activity is detectable in normal cervical epithelium, and that an gradual increase exists for both telomerase activity and HPV positivity from normal epithelium to HGSIL. However, HPV infection and telomerase activity appear to be independent of each other. The high frequency of telomerase positivity in patients with normal cervical epithelium indicates that telomerase activity is not a useful differential diagnostic aid. Whether patients with telomerase-positive dysplasias have a higher probability to progress into an invasive carcinoma remains to be clarified by follow-up studies.     

Autofluorescence spectroscopy of malpighian epithelial cells,as a new tool for analysis of cervical cancer precursors

A spectroscopic analysis of autofluorescence was investigated within the cell cytoplasm from cervical malpighian epithelia prepared on Thin-Prep smears. Autofluorescence emission spectra from 22 cervix were analyzed by microspectrofluorometry under a 363 nm laser excitation. Among the analyzed cervix, 6 were in normal limits, 6 in inflammatory limits, 5 were evocative of Low-Grade Squamous Intraepithelial Lesions (LGSILs) and 5 were evocative of High-Grade Squamous Intraepithelial Lesions (HGSILs). Cytoplasmic emission intensities at 450 nm of cells from inflammatory, LGSIL and HGSIL cervix were equivalent and were 3-fold higher than from normal cervix. All smears presented a two-fold lower autofluorescence emission in the cytoplasm than in the nucleus. The spectral profile analysis allows the discrimination of cells from inflammatory, LGSIL and HGSIL cervix. The 525/425 nm emission ratios were 0.75+/-0.1, 0.96+/-0.04 and 1.2+/-0.1 for inflammatory, LGSIL and HGSIL, respectively. We suggest that smears of normal, inflammatory, LGSIL and HGSIL cervix could be discriminated by the analysis of the 450 nm emission intensity and 525/425 nm emission ratios from cells of malpighian epithelia.     

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.