Telomerase activity in candidate stem cells from fetal liver and adult bone marrow

Telomerase is a ribonucleoprotein polymerase that synthesizes telomeric repeats onto the 3' ends of eukaryotic chromosomes. Activation of telomerase may prevent telomeric shortening and correlates with cell immortality in the germline and certain tumor cells. Candidate hematopoietic stem cells (HSC) from adult bone marrow express low levels of telomerase, which is upregulated with proliferation and/or differentiation. To address this issue, we stimulated purified candidate HSC from human adult bone marrow with stem cell factor (SCF), interleukin-3 (IL-3), and Flt3-ligand (FL). After 5 days in culture, activity was detected in total cell extracts from IL-3-, SCF + FL-, SCF + IL-3-, FL + IL-3-, and SCF + IL-3 + FL-stimulated cultures, but not from cells cultured in SCF or FL alone. Within the CD34(+) fraction of the cultured cells, significant activity was found in the CD34(+)CD71(+) fraction. In addition, PKH26 staining confirmed that detectable telomerase activity was present in dividing PKH26(lo) cells, whereas nondividing PKH26(hi) cells were telomerase negative. Because in these experiments no distinction could be made between cycling "candidate" stem cells that had retained or had lost self-renewal properties, fetal liver cells with a CD34(+)CD38(-) phenotype, highly enriched for cycling stem cells, were also examined and found to express readily detectable levels of telomerase activity. Given the replication-dependent loss of telomeric DNA in hematopoietic cells, these observations suggest that the observed telomerase activity in candidate stem cells is either expressed in a minor subset of stem cells or, more likely, is not sufficient to prevent telomere shortening.     

Effects of castration on apoptosis and transforming growth factor-beta 1 mRNA in the neonatal mouse seminal vesicles

BACKGROUND: In the adult male rat prostate, castration induces apoptosis of epithelial cells concomitant with the increase in transforming growth factor-beta 1 (TGF-beta 1). In the present study, we investigated the effects of castration on apoptosis and TGF-beta 1 mRNA in neonatal mouse seminal vesicles. METHODS: 5-day-old BALB/c mice were castrated by Pfeifer's method. We estimated the weight, 3H-thymidine uptake by whole seminal vesicles, the amount of TGF-beta 1 mRNA by RT-PCR method, and the apoptotic index of both epithelium and mesenchyme. RESULTS: The castration of 5-day-old neonatal mice resulted in much less weight of seminal vesicles and DNA synthesis estimated by 3H-thymidine uptake by whole seminal vesicles compared to intact neonatal mice, indicating that the growth of neonatal mouse seminal vesicles depends on androgens secreted by the testis. The amount of TGF-beta 1 mRNA estimated by RT-PCR method increased 4 days after castration at 5 days of age. However, the castration did not induce apoptosis in the seminal vesicles. CONCLUSION: The present study indicates that castration of neonatal mice does not induce apoptosis in the seminal vesicles, although it does a transient increase in TGF-beta 1 mRNA in the seminal vesicles.     

Telomerase inhibitor

Human normal somatic cells and tissues have undetectable or very weak telomerase activity and shorten telomere size at each cell division resulting in a limited proliferative life span. Human germ tissues and most tumor tissues have telomerase activity and maintain telomere size during cell proliferation resulting in unlimited growth. Telomerase is expected to be a new target for cancer chemotherapy. Cultured tumor cells are shown to die after losing telomerase activity.     

Calreticulin: an intracellular Ca++-binding protein abundantly expressed and regulated by androgen in prostatic epithelial cells

Calreticulin was identified in a screen for androgen-response genes in the rat ventral prostate. Northern blot and Western blot analyses in the rat model showed that both calreticulin messenger RNA and protein are down-regulated by castration and up-regulated by androgen replacement in the prostate. Northern blot analysis showed that calreticulin expression level in the prostate is much higher than that in seminal vesicles, heart, brain, muscle, kidney, and liver. The regulation of calreticulin expression by androgen is only observed in the prostate and seminal vesicles, two male secondary sex organs. The induction of calreticulin by androgen in prostate organ culture partially resists protein synthesis inhibition, suggesting that calreticulin is a direct androgen-response gene. In situ hybridization and immunohistochemistry studies showed that calreticulin is an intracellular protein in prostatic epithelial cells. Because calreticulin is a major intracellular Ca++-binding protein with 1 high-affinity and 25 low-affinity Ca binding sites, our observations suggest that calreticulin is a promising candidate that mediates androgen regulation of intracellular Ca++ levels and/or signals in prostatic epithelial cells. The expression of calreticulin is also regulated by androgen in the mouse and human prostate, suggesting that androgen regulation and function of calreticulin in the prostate are conserved evolutionarily.     

Telomerase activity of sarcoma cell lines and fibroblasts is independent of p53 status

Telomerase activity is necessary for the stabilization of telomeres, which function to overcome cellular senescence and are linked to unlimited cell proliferation. Activation of telomerase is characteristic of immortalized cell lines and most tumors. The p53 gene has been implicated as a crucial barrier to unlimited cell proliferation, and its absence has been shown to allow direct immortalization of cells by certain oncogenes. The p53 gene may have an additional function of signaling cell growth arrest in response to telomere shortening, which occurs with repeated cellular divisions and ultimately threatens chromosomal stability. This prompted us to consider whether the enzyme telomerase, responsible for adding new telomeres to chromosomal ends, may be affected by the p53 status of normal and malignant cells. We investigated whether a relationship between telomerase and p53 could be demonstrated in a human sarcoma cell line containing a missense p53 mutation and several stable transfectants that express the wild-type p53 gene or a temperature-sensitive mutant of p53. All cell lines had readily detectable telomerase activity regardless of p53 status. In addition, murine fibroblast cell strains established from tissues of p53+/+ and p53-/- (p53 knockout) mice expressed telomerase regardless of the p53 status of their tissue of origin. Levels of telomerase subunit mRNA (hEST2) were comparable among cell lines and tissues with different p53 status. These results imply that p53 status is not associated with telomerase activity per se and that activation of telomerase can occur either in cells completely devoid of p53 or in cells that have functional p53.     

Primary small cell anaplastic carcinoma of the breast diagnosed by fine needle aspiration cytology: a case report

Telomerase, a ribonucleoprotein that adds telomeric repeats onto chromosome ends, is involved in telomere length maintenance and permits unlimited cell proliferation. We examined the possibility that higher telomerase activity is associated with the replicative phase of the cell cycle using gastric cancer cell lines treated with anticancer drugs. Telomerase activity increased at the time point of S-phase accumulation in NUGC-3 cells (5 x 10(5) cells/ml) incubated with CDDP (0.5 microgram/ml), paclitaxel (0.01 microM), or VP-16 (1 microM) and in MKN-28 cells incubated with CDDP. When these cell lines were incubated with 5-fluorouracil (10 microM) or CPT (0.1 microM), the increase of telomerase activity preceded the S-phase accumulation. Our results suggest that telomerase activity be regulated by the cell cycle.     

High incidence and histogenesis of seminal vesicle adenocarcinoma and lower incidence of prostate carcinomas in the Lobund-Wistar prostate cancer rat model using N-nitrosomethylurea and testosterone

The origin of chemically induced male accessory sex gland tumors was studied in Lobund-Wistar rats. Rats were treated at the age of 3 months with a single intravenous injection of 30 mg N-nitrosomethylurea (NMU)/kg body weight and given subcutaneous silastic implants filled with 40 mg testosterone propionate. Previous reports described a high incidence of prostate carcinomas in these rats with this treatment protocol. Additional animal groups included untreated controls, rats that received only an injection of 30 mg NMU/kg, and rats that were subjected to ablation of the seminal vesicle lobes prior to the treatment with NMU and testosterone. Three to 14 rats per group were sacrificed 4 to 10 months after NMU treatment and all remaining rats after 12 months. Twenty-four additional rats died or became moribund during the study. All rats were necropsied and the dorsolateral and ventral prostate and seminal vesicles with coagulating gland (anterior prostate) were examined histologically according to a standardized protocol. Lesions detected included atypical hyperplasia in all glands (resembling prostate intraepithelial neoplasia of human beings), adenomas in seminal vesicles only, and early carcinomas and adenocarcinomas in seminal vesicles and coagulating gland. Early carcinomas of the seminal vesicle, microscopically small and with invasion of the lamina propria and/or tunica muscularis, were detected as rapidly as 4 months after treatment. The vast majority (> 95%) of the grossly visible nodules/masses originated from the seminal vesicles. Testosterone treatment enhanced occurrence and increased the incidence of all lesions, particularly of seminal vesicle adenocarcinomas, from 30% (7/23) to 64% (21/33). Coagulating gland tumors were found in 21% (7/33) of the rats. Ablation of the seminal vesicle lobes reduced the incidence of seminal vesicle adenocarcinomas to 11% (3/29), and these tumors arose from tissues remaining within the parenchyma of the seminal vesicle/prostate complex after ablation. Thus, NMU-induced and testosterone-promoted male sex gland tumors of the Lobund-Wistar rat arise almost exclusively in the seminal vesicles and coagulating gland (anterior prostate), are highly invasive in seminal vesicles before attaining a grossly visible size, and progress rapidly within 4 months, spreading to adjacent tissues and other organs.     

Scanning electron microscopy of the accessory sex glands of the adult male rat

A systematic, comparative study of the accessory sex glands of the adult male rat after androgen withdrawal was carried out. The changes were investigated by using scanning electron microscopy at different intervals after surgical castration. The main common signs of epithelial cell involution were flattening of the cell surface, reduction of the size and number of microvilli, some blurring of the cell borders, cessation of secretory activity and diminution of the luminal volume of the glands. Overall, confident signs of atrophy were evident after one week, and complete epithelial involution was reached by the third week. The epithelial cell atrophy was accompanied by a relative stromal hyperplasia. The new observation seems to be that the process of stroma consolidation is progressing for a considerable time subsequent to the completion of the epithelial involution. This phenomenon is particularly evident in the dorsal prostate, the seminal vesicle and the coagulating gland.     

Multiple pathways to cellular senescence: role of telomerase repressors

Telomeres progressively shorten with age in somatic cells in culture and in vivo because DNA replication results in the loss of sequences at the 5' ends of double-stranded DNA. Whereas somatic cells do not express the enzyme, telomerase, which adds repeated telomere sequences to chromosome ends, telomerase activity is detected in immortalised and tumour cells in vitro and in primary tumour tissues. This represents an important difference between normal cells and cancer cells, suggesting that telomere shortening causes cellular senescence. Hybrids between immortal cells and normal cells senesce, indicating that immortal cells have lost, mutated or inactivated genes that are required for the programme of senescence in normal cells. Genes involved in the senescence programme have been mapped to over ten different genetic loci using microcell fusion to introduce human chromosomes and restore the senescence programme. Multiple pathways of cellular senescence have also been demonstrated by chromosome transfer, indicating that the functions of the mapped senescence genes are probably different. One possibility is that one or more of these senescence genes may suppress telomerase activity in immortal cells, resulting in telomere shortening and cellular senescence. To test this hypothesis, telomerase activity and the length of terminal restriction fragments (TRFs) have been examined in microcell hybrids. Re-introduction of a normal chromosome 3 into the renal cell carcinoma cell line RCC23, which has the short arm of chromosome 3, restored cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells containing the introduced chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to the parental RCC23. Thus, restoration of cellular senescence by chromosome 3 is associated with repression of telomerase function in RCC cells. This evidence suggests that telomerase suppression is one of several pathways involved in immortalisation.     

Epidural anaesthesia for caesarean section in an achondroplastic dwarf

Telomerase is a ribonucleoprotein that synthesizes telomeric DNA on chromosomal ends. Telomerase activation has been seen in many immortal cell lines and cancers. Telomerase activity was analyzed in prostate carcinoma; in coexistent prostatic intraepithelial neoplasia (PIN), benign prostatic hyperplasia (BPH), atrophy and normal tissue; and in benign prostate glands. Telomerase activity was detected in 80 of 87 (92%) prostate cancers. Forty-one matched samples (from a total of 32 cases) were available for comparative analysis. The presence of telomerase activity in adjacent PIN, BPH, and normal tissue was correlated with telomerase activity in the malignant epithelium. In these adjacent tissues, telomerase activity was found in 11 of 15 (73%) PINs, 13 of 26 (50%) BPHs, and 1 of 6 (16%) atrophy and 4 of 11 (36%) normal tissues. In contrast to the BPH tissue from cancer-bearing glands, all 16 BPH specimens from patients only diagnosed with BPH were telomerase activity negative. In cancer samples, there was no correlation between telomerase activity and Gleason grade or preoperation prostate-specific antigen level. Our data indicate that telomerase activity is present in most prostate cancers. The high rate of telomerase activity in the benign-appearing areas of these glands may be attributed either to the presence of occult cancer cells or to early molecular alterations of cancer that were histologically inapparent.     

Ultrastructure and lectin cytochemistry of the cloacal pelvic glands in the male newt Triturus marmoratus marmoratus

The cloacal organ of Salamandridae species contains four glands: pelvic, dorsal, ventral, and Kingsbury's glands. Pelvic glands have been studied only by light microscopy with conventional methods, and consist of multiple tubular serous glands with a prismatic epithelium which contains numerous PAS positive secretory granules. The present report is an ultrastructural and lectin cytochemistry characterization of the pelvic glands of Triturus marmoratus marmoratus throughout the reproductive cycle. Our methods consisted of conventional electron microscopy, and colloidal-gold lectin cytochemistry of the following lectins: WGA, ConA, LcA, UEA-I, PNA, SBA, and HPA. In the prereproductive period, the glands showed a tall epithelium which consisted of two cell types, dark and clear cells, surrounded by elongated, myoepithelial cells. Both dark and clear cells showed the ultrastructural characteristics of secretory cells, and exhibited many secretory granules in the apical cytoplasm. Areas showing densely packed, degenerating cell organelles--which were not surrounded by membrane--were observed in the dark cells whereas the clear cells showed large heterolysosomes. In the postreproductive period the number of secretory granules decreased, the rough endoplasmic reticulum was less developed, and areas of degenerating organelles were absent. In addition, small basal cells appeared. The results of the lectin histochemistry study were similar in both reproductive periods. In the epithelial cells, the rough endoplasmic reticulum, the Golgi complex, and secretory granules exclusively labeled to ConA. In all cell types, the nuclei reacted to all lectins while the cytosol only reacted to LcA lectin. The ultrastructural and histochemical characteristics of the pelvic glands of T. marmoratus suggest that these glands could be homologous to the mammalian seminal vesicles and prostate.     

Apnea following spinal anaesthesia in two former pre-term infants

Telomerase activity is associated with the proliferative activity of cells. In the endometrium, telomerase activity is higher in the proliferative phase than in the secretory phase of the menstrual cycle, suggesting that telomerase activity may occur primarily in the glandular epithelial cells. To test this, a dissociated cell culture of the endometrium was performed, and the telomerase activity in each cell fraction was analysed. Telomerase activity was found in all 10 endometrial tissues of the proliferative phase of the menstrual cycle. Both the fragments of epithelial glands and single cells, which were prepared by enzymatic dissociation, showed telomerase activity. In the 7 day cell culture, it was found in nine out of 10 epithelial cell enriched fractions, but in none of the stromal cell enriched fractions. Flow cytometric analysis showed that the epithelial enriched fraction was contaminated with a predominant number of stromal cells, while the stromal cell enriched fraction was comprised mostly of stromal cells with apparent proliferative activity. Our results suggest that telomerase activity of the endometrium occurs primarily in the epithelial cells in the endometrium and that the stromal cells do not express telomerase activity regardless of their potent proliferative activity.     

Changing patterns of gene expression identify multiple steps during regression of rat prostate in vivo

The rat ventral prostate is an androgen-dependent organ that undergoes dramatic cell death upon removal of testosterone by surgical castration. Several well characterized criteria, such as nuclear condensation, organelle blebbing, and DNA fragmentation, have been used to demonstrate that most of this cell loss is due to programmed cell death, or apoptosis, of the secretory epithelial cells. In addition to changes in morphology, it is well known that cells undergoing apoptosis show alterations in gene expression, and it is widely assumed that many of these genes are directly involved in the mechanism of programmed cell death. Using poly A+ RNA derived from normal rat prostate as well as from the regressing prostates of castrated rats, we have used a PCR-based subtractive hybridization approach to generate complementary DNA (cDNA) libraries greatly enriched in cDNAs strongly regulated during rat prostate regression. Several hundred of the genes represented in these libraries appear to be strongly regulated during prostate regression and most of these are prostate specific. Sequence analysis indicates that up to 30% of these clones are similar or identical to genes of known function, approximately 20% are similar to expressed sequence tags (ESTs), and as many as 50% of these clones have not been characterized previously. Analysis of selected clones using in situ hybridization indicates that they are expressed specifically in prostate epithelial cells, and that certain of these clones are regulated temporally in a pattern consistent with apoptosis. The patterns of gene expression include: 1) genes whose expression decreases uniformly after removal of androgen, indicative of androgen sensitive genes; 2) genes whose expression increases in apoptotic prostate cells and in other tissues, suggesting a class of genes generally involved in apoptosis; 3) and genes whose expression increases in individual regressing prostate epithelial cells, suggesting a class of prostate specific genes associated with apoptosis.     

Telomerase activity in the normal and neoplastic rat mammary gland

The 1-methyl-1-nitrosourea-induced rat mammary tumor model system is well studied, reproducible, and widely used. We have investigated whether these tumors possess higher telomerase activity than normal mammary tissue. Using the telomeric repeat amplification protocol assay, we found significantly higher telomerase activity in 36 mammary carcinomas than in 72 mammary glands of virgin rats. The level of telomerase activity in virgin rats was unaffected by strain, age, stage of the estrous cycle, or ovariectomy. However, mammary glands obtained from pregnant rats exhibited telomerase activity comparable to that found in the tumors, possibly reflecting the high epithelial content of these tissues. Indeed, isolated epithelial cells from virgin and pregnant mammary glands and from carcinomas had similar telomerase activities. Thus, telomerase activity is constitutive in the rat mammary epithelium and is not a unique characteristic of malignant transformation in this tissue. These results underscore the importance of attributing biochemical properties to specific cell types in a tissue, a situation not paralleled in the interpretation of data from in vitro models.     

Regional expression of transforming growth factor-alpha in rat ventral prostate during postnatal development, after androgen ablation, and after androgen replacement

The prostate is a highly heterogeneous organ, composed of different types of epithelial and stromal cells organized regionally along the ductal network. Although androgen-stimulated growth and maintenance of the prostate gland primarily involve epithelial cells, it is unclear whether all epithelial cells are androgen dependent. Moreover, the actions of androgens may not be direct; a number of polypeptide growth factors, including transforming growth factor-alpha (TGFalpha), are postulated to mediate androgen action in the rat prostate. In this investigation, using an immunohistochemical technique, we examined the cellular and regional expression of TGFalpha in the rat ventral prostate during postnatal development to adulthood. TGFalpha-immunopositive cells were located throughout the ductal epithelium from postnatal days 5-20. By day 45 and thereafter, regional variation in TGFalpha expression became apparent; epithelial cells in the proximal segment exhibited intense staining, whereas those in the distal segment exhibited negligible staining. These observations were coincident with increased serum testosterone concentrations at puberty. To understand the role of androgen in the expression of TGFalpha in the epithelial cells of the distal and proximal segments of the adult rat ventral prostate, androgen was withdrawn by castration, and testosterone subsequently was administered. Androgen receptor protein expression decreased after castration and reappeared after androgen replacement in both the distal and proximal segments. TGFalpha staining was negligible in epithelial cells of the distal segment of intact adult rats, became prominent by 7 days after castration, but then diminished after the administration of testosterone. Western blot analyses revealed the presence of a specific 30-kDa immunoreactive form of TGFalpha in rat ventral prostate, and its quantity reflected the staining intensities observed in the immunohistochemical studies. These results suggest that TGFalpha expression is negatively regulated by androgen in epithelial cells of the distal segment. In contrast, staining for TGFalpha in epithelial cells of the proximal segment did not change with castration or testosterone administration, suggesting that TGFalpha is not regulated by androgen in this region of the ventral prostate. In summary, TGFalpha expression is differentially regulated among epithelial cells localized in two different regions of the ventral prostate. We hypothesize that TGFalpha may function as a survival factor for epithelial cells which, as a consequence of its expression, become androgen independent and thus escape apoptotic cell death after androgen ablation.     

Effects of hypoxemia on the electroretinogram in diabetics

The localization of the neural cell adhesion molecule L1 in the male urogenital tract (including seminal vesicles and prostate) of the mouse and bull was investigated using immunocytochemical and immunochemical methods in order to better understand the function of this glycoprotein in non-neural tissues. L1 antibodies labeled non-myelinated nerves in all portions of the urogenital tract investigated. However, L1 immunoreactivity was also found between epithelial cells of several regions of the urogenital system including epididymal tail, deferent duct, ejaculatory duct and seminal vesicles. Some L1 immunoreactivity was also demonstrated between epithelial cells of murine urinary bladder and urethra. The specificity of the immunoreaction was verified by western blots. There was no correlation between L1 expression and proliferating activity as revealed by double immunocytochemistry using various markers of cell proliferation. This unexpected expression of L1 in nonneural tissues is mainly restricted to non-proliferating epithelia of those portions of the urogenital tract that are derived from the Wolffian duct. It is suggested that L1 in these epithelia could enhance the mechanical resistance and reduce transepithelial permeability.     

Telomerase activity in skeletal sarcomas

BACKGROUND: Telomerase is a ribonucleoprotein that adds TTAGGG nucleotide repeats onto the ends of eukaryotic chromosomes to maintain telomere integrity. Somatic cells do not express telomerase and stop dividing when the chromosomal ends are shortened critically after many cell divisions. Immortal cell lines and cancer cells apparently have telomerase activity that contributes to an unlimited number of cell cycles. The purpose of our study is to investigate whether telomerase activity is expressed in primary malignant tumors of the skeletal system when compared to adjacent normal tissue. METHODS: Fresh tumor and normal tissue was collected from 14 patients (10 males, 4 females; age range, 8 to 76 years) and protein extraction performed. The tumors included seven osteosarcomas (three examined before and after chemotherapy), two chondrosarcomas, two spindle cell tumors, one hemangiopericytoma, one chordoma, and one adamantinoma. Telomerase activity was analyzed by using a highly sensitive polymerase chain reaction (PCR)-based assay (telomere repeat amplification protocol [TRAP]). RESULTS: Telomerase activity was found in 8 of 14 sarcoma patients (57%) using the TRAP assay. Compared to HeLa cell extract (positive control), telomerase activity in the tumor specimen ranged from 0 (in osteosarcoma) to 11.7% (in hemangiopericytoma). There was variation in the number of telomeric repeats generated by telomerase. At least five telomeric bands (e.g. 50, 56, 62, 68, 74 bp) in a ladder pattern had to be present before telomerase activity was considered positive in our analysis. CONCLUSIONS: Telomerase activity may be an oncogenic sustaining event helping to maintain the transformed phenotype seen in malignant tumors of the bone. The degree of telomerase activity varies among skeletal malignancies, but was less than that observed in HeLa cells. The majority of osteosarcomas showed no telomerase activity.     

Castration-induced apoptosis in the rat ventral prostate is associated with increased expression of genes encoding insulin-like growth factor binding proteins 2,3,4 and 5

Insulin-like growth factor binding proteins (IGFBPs) have recently been demonstrated to act as regulators of apoptosis in vitro in both prostate and breast cancer cell lines. We show here that gene expression of IGFBP-2,-3,-4 and -5 increase rapidly in the rat ventral prostate following castration. Increases in IGFBP mRNA levels were detectable by Northern blotting by 6 hours and reached 5 to 10 fold of control levels at 72 hours after castration. Apoptosis in the ventral prostate, as detected in situ by the TUNEL method, was also induced as early as 6 hours after castration. TRPM-2/clusterin, a gene known to be associated with involution of the prostate, was not detected in sham castrated controls but was expressed by 24 hours following androgen ablation. IGF-I mRNA levels increased to 160% of control values within 6 hours following castration, then decreased gradually over the next 72 hours to 35% of control. Affinity labelling experiments demonstrated that IGF-I receptor levels increased initially after castration with peak binding at 24 hours, then declined to levels lower than control. These results suggest that rapid induction of IGFBPs in the rat ventral prostate following androgen ablation may play a role in apoptosis and involution of the prostate gland.