Localization of telomerase hTERT protein and hTR in benign mucosa, dysplasia, and squamous cell carcinoma of the cervix.

Telomerase has been detected by telomerase repeat amplification protocol (TRAP) assay in cervical dysplasia and squamous cell carcinoma but not in most normal cervical tissues. In the present study, the cellular localization of the protein catalytic subunit of telomerase (hTERT) and the RNA component (hTR) were investigated by a sensitive immunohistochemical technique and by in situ hybridization, respectively. hTERT protein was detected in all diagnostic categories of cervical specimens. hTERT was localized predominantly to the lower suprabasal levels of normal squamous mucosa but was detected throughout virtually all levels of the lesional epithelium in low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs), and squamous cell carcinoma (SCC). Telomerase expression correlated with hTERT detection in SCC and HSIL but was not detected by TRAP assay in most samples of normal mucosa or LSIL. The distribution of hTR correlated with the localization of hTERT in HSIL and SCC but was restricted to the basal and suprabasal cell layers in normal mucosa and LSIL.

Quantitative telomerase expression in glioblastomas shows regional variation and down-regulation with therapy but no correlation with patient outcome.

Despite the nearly ubiquitous expression of telomerase in almost all types of malignant human tumors, studies have shown widely varying positivity in the highest-grade glioma, the glioblastomas (GBMs), ranging from 26% to 100% of tumors analyzed. We have previously shown significant variability in positive versus negative telomerase expression from region to region within the same GBM. In this study, we hypothesized that application of new quantitative methodology would extend our previous observations and identify whether there is heterogeneity in levels of protein expression even within areas positive for telomerase in high-grade gliomas. Finally, we sought to correlate quantitative telomerase expression with patient outcome and therapeutic response. Quantitative analysis was achieved by polymerase chain-based TRAP assay with phosphorimager analysis and compared with clinical information obtained from 19 patients, most with primary, untreated GBMs. Results showed up to 3-fold variability in telomerase levels across multiple regional samples from the same patient, as well as between patients. In 5 of 6 patients with recurrent tumors who had received intervening radiation therapy or chemotherapy, telomerase was downregulated in the second, post-therapy sample. These data provide in vivo corroboration of recent in vitro experiments showing telomerase downregulation after radiation therapy or chemotherapy treatment of cell lines. Our finding of variability in levels of telomerase expression in GBMs parallels the known heterogeneity of these tumors for histologic features and cell growth-related factors. Statistical analysis showed no relationship between TRAP score and either time to clinical progression or time to death.

Comparison of telomerase activity and GSTP1 promoter methylation in ejaculate as potential screening tests for prostate cancer.

New diagnostic tools are needed for the early detection of prostatic cancer. The molecular detection of prostate cancer cells in ejaculates was evaluated using complementary PCR-based methods. LNCaP cells, a cell line derived from prostatic carcinoma, were spiked into normal seminal ejaculates and the prostatic epithelial component of the specimens was isolated by immunomagnetic bead sorting, using a monoclonal antibody to prostate-specific membrane antigen (PSMA). Ejaculates from nine patients with a recent diagnosis of prostate cancer were processed in a similar fashion, using LNCaP-spiked aliquots as an internal positive control. Telomerase expression was evaluated by the telomeric repeat amplification protocol (TRAP) and glutathione S-transferase gene promoter (GSTP1) hypermethylation was evaluated by methylation-sensitive restriction endonuclease digestion and PCR amplification. Telomerase activity was detected in LNCaP cells recovered from normal seminal ejaculates but was not found in all nine samples from patients with prostate cancer. The sensitivity of GSTP1 analysis was similar to telomerase analysis for the detection of LNCaP cells from normal ejaculate samples but was positive in ejaculates from four out of nine patients with prostate cancer. GSTP1 DNA methylation status is more sensitive than telomerase analysis for the detection of malignant cells in seminal ejaculates from patients with prostate cancer.

Dehydration-associated changes in the ventral glial limitans subjacent to the supraoptic nucleus include a reduction in the extent of the basal lamina but not astrocytic process shrinkage.

In these studies we have investigated factors that might account for two previous observations of the ventral glial limitans subjacent to the supraoptic nucleus (SON-VGL) of dehydrated rats: (1) a reversible reduction in the thickness of the SON-VGL, and (2) a reversible reorientation of VGL astrocytes. Since components of the basal lamina influence both cell viability and polarity, we used electron microscopic sterology to determine the volume fraction of basal lamina in the SON-VGL. We further made extensive measurements of astrocytic process thickness to determine if cellular shrinkage is a factor in the thinning of the SON-VGL. While we found no evidence for changes in the thickness of astrocytic processes, there was a significant and reversible reduction in the extent of the basal lamina. These data suggest that the thinning of the VGL is due to complex biochemical events and is not merely an epiphenomenon of dehydration.

Plasticity of astrocytes of the ventral glial limitans subjacent to the supraoptic nucleus.

We present evidence of gross morphological changes in astrocytes of the ventral glial limitans (VGL) associated with a well-known model of central nervous system (CNS) plasticity: the hypothalamic supraoptic nucleus (SON). Activity of SON magnocellular neuroendocrine cells (MNCs) was stimulated in experimental rats by substitution of 2% saline for drinking water for 2 or 9 days. Light microscopic measures revealed that a significant decrease in VGL thickness, by 34%, occurred with 9 days of stimulation. Astrocyte nuclei of 9-day dehydrated animals were also found to be 39% closer to the pial surface when compared with controls. Electron microscopy revealed a reorientation of individual astrocytes from a direction perpendicular (vertical) to the pial surface, to one parallel (horizontal) to this region. Vertically oriented astrocytes were found to be greater in the control group, by 49%, when compared with the 9-day dehydrated group, where cells were predominantly horizontal in orientation. Vertically oriented cells were further analyzed as to the direction of their vertical projections. Control, 2-day dehydrated and 9-day rehydrated animals, had more vertical cells which were oriented toward the pial surface when compared with 9-day dehydrated animals, where the relatively few vertically oriented astrocytes were significantly more likely to project toward the dendritic zone. In animals allowed to rehydrate for 9 days following a period of dehydration, these changes returned toward control levels. We conclude that astrocytes in vivo are capable of reversible gross morphological changes over a relatively short time.