Reversible downregulation of telomerase activity by hyperthermia in osteosarcoma cells

Telomerase, a ribonucleoprotein enzyme, maintains telomere length and is expressed by the majority of malignant tumours, but not in normal tissue. Telomerase facilitates the division of tumour cells and its activity has been suggested as a prognostic indicator, but so far the regulation or modulation of telomerase activity has not been described. Hyperthermia has been shown to decrease tumour growth by inhibition of proliferation. Therefore, the effect of hyperthermia on telomerase activity in human osteosarcoma cells was studied. Telomerase activity was measured by the Telomeric Repeat Amplification Protocol (TRAP) assay in three different osteosarcoma cell lines subjected to hyperthermia (42.5°C, 90min) and in controls cultured under basal conditions (37°C). Telomerase activity was strongly inhibited by hyperthermia and decreased in all cell lines tested after a recovery time of 2h under basal conditions (37°C) to an activity of 85%, after 12h 60% and with lowest activity 55% compared to activity of control cells. Telomearase activity then increased and reached the same, i.e. basal, level as before hyperthermia, after 112 h. These results show that hyperthermia results in a reversible downregulation of telomerase activity in osteosarcoma cells. This effect facilities studies on the regulation of telomerase activity and detailed information might lead to new therapeutic strategies.     

Apoptosis induced by hyperthermia in Dunn osteosarcoma cell line in vitro.

The effect of hyperthermia at 43.5 degrees C for 1 h on Dunn osteosarcoma cells was studied. With sham-heated cells (37 degrees C, 1 h) as the control, the hyperthermia treated cells were divided into five groups. Time 0 group was the cells that were harvested immediately after heated at 43.5 degrees C for 1 h. Whereas time 3, 6, 12, and 24 h groups were the cells that were collected respectively after reincubation at 37 degrees C for the above different time periods. The appearance of hyperthermia-induced apoptosis of Dunn osteosarcoma cells was demonstrated to be time dependent. With the confocal microscopic study and TUNEL staining, the morphological characteristics of apoptosis, condensed nuclei and fragmented nuclei were obvious when reincubated at 37 degrees C for 6 h after hyperthermic treatment. This hyperthermia-induced apoptosis was further confirmed by flow cytometric analysis on DNA contents. The sub-G1 region that was proposed as a marker of apoptotic cells was most significantly elevated at 6 h after hyperthermic treatment and, thereafter, decreased to the levels of control values by 24 h, as the apoptotic cells underwent secondary necrosis and degraded to debris. The DNA strand breaks, considered as the key biochemical event of apoptosis, were detected by the TUNEL assay. This study indicated that hyperthermia (43.5 degrees C for 1 h) can induce apoptotic changes on osteosarcoma cells in vitro very rapidly (within 6 h after treatment), and its occurrence might not be detected if the samples are not taken at several early time points after hyperthermia.     

恶性肿瘤细胞体外诱导逆转过程中端粒酶活性变化的研究

目的 观察骨肉瘤细胞株HOS和神经母细胞瘤细胞株Neuro-2A在体外诱导逆转中端粒酶活性的变化。探讨端粒酶活性作为恶性肿瘤细胞逆转指标的意义。方法 体外用维甲酸（RA）和1，7-二羟夹氧蒽酮（DX-1）分别对HOS和Neuro-2A细胞进行逆转诱导，观察处理后瘤细胞的形态和功能变化，并采用TRAP-银染法检测诱导逆转过程中瘤细胞端粒酶活性的改变。结果 经诱导剂处理后，HOS和Nenro-2A细胞生长均受到明显抑制，形态和功能检测显示细胞出现逆转改变，HOS细胞的端粒酶活性随诱导剂作用时间的延长及其浓度的增加明显减弱，Neuro-2A细胞株在中，低剂量诱导剂作用下。端粒酶活性无明显下降，仅高剂量组端凿酶活性明显下降。结论 HOS和Neuro-2A细胞株在体外诱导恶性逆转的过程中，瘤细胞形态和功能均出现明显改变。端粒酶活性的下调可作为部分恶性肿瘤细胞恶性逆转的指标之一。     

Superoxide dismutase levels in Chinese hamster ovary cells and ovarian carcinoma cells after hyperthermia or exposure to cycloheximide

Superoxide dismutase (SOD) activity in Chinese hamster ovary (CHO) and ovarian carcinoma (OvCa) cells was measured after exposure to hyperthermia and correlated with the development of thermotolerance. The SOD activity of each cell type was largely copper- and zinc-containing SOD activity. Both cell types had similar but low levels of SOD activity when the cells were grown at 37 degrees C. After exposure for 2 h at 41.5 degrees C, SOD activity of OvCa cells, but not of CHO cells, was increased. After exposure to 45 degrees C for 15 min, SOD activity was also increased in the OvCa cells, but not in CHO cells. After 15 min at 45 degrees C followed by 1 h incubation at 37 degrees C, SOD activity was increased in OvCa and CHO cells; after an 8-h incubation at 37 degrees C, SOD activity doubled in each cell type. Thermotolerance is maximal after 2 to 3 h of exposure at 41.5 degrees C and after 8 to 10 h incubation at 37 degrees C following exposure to 15 min at 45 degrees C. The turnover of SOD activity in OvCa cells was estimated by the rate at which activity was lost following addition of cycloheximide (10 micrograms/ml). Twenty-four % of the activity was lost with a half-life of 10 min, and 76% was lost with a half-life of 4.5 h. Despite restriction of general protein synthesis 3 to 4 h after 45 degrees C hyperthermia, SOD activity was increased at 1 and 8 h after exposure, presumably coincidental with heat shock protein synthesis and development of thermotolerance. These data suggest that SOD activity may be important in protecting cells exposed to heat and that it may play a role in the development of thermotolerance.     

SV40 infection induces telomerase activity in human mesothelial cells

Mesotheliomas are malignant tumors of the pleural and peritoneal membranes which are often associated with asbestos exposure and with Simian virus 40 (SV40) infection. Telomerase activity is repressed in somatic cells and tissues but is activated in immortal and malignant cells. We evaluated telomerase activity in seven primary malignant mesothelioma biopsies and matched lung specimens and 20 mesothelioma cell lines and eight corresponding primary tumor cultures. All the tumor biopsies, and nearly all primary cell mesothelioma cultures and cell lines were telomerase positive. The findings in cell lines paralleled those observed in primary cultures in cases where paired samples were available. Next, we found that SV40, a DNA tumor virus present in approximately 50% of mesothelioma biopsies in the USA, induced telomerase activity in primary human mesothelial cells, but not in primary fibroblasts. Telomerase activity became detectable as early as 72 h following wild-type (strain 776) SV40 infection, and a clear DNA ladder was detectable 1 week after infection. The amount of telomerase activity increased during passage in cell culture and appeared to parallel increases in the cellular amounts of the SV40 large T-antigen. Thus, SV40 infection leads to telomerase activity before the infected mesothelial cells become transformed and immortalized. SV40 infection of human fibroblasts did not cause detectable telomerase activity. We also determined that the SV40 small t-antigen (tag) plays an important role in inducing telomerase activity because this activity was undetectable or minimal in mesothelial cells infected and/or transformed by SV40 tag mutants. Asbestos alone did not induce telomerase activity, and asbestos did not influence telomerase activity in mesothelial cells infected with SV40. Induction of telomerase activity by SV40 may be related to the very high rate of mesothelial cell immortalization that is characteristically associated with SV40 infection of mesothelial cells.     

Telomerase activation in nasopharyngeal carcinomas.

Nasopharyngeal carcinomas (NPC) are common in Hong Kong and southern China but rare in Western countries. Telomerase activation is common in human cancers but has not been reported previously in NPC. Telomerase activation in NPC was determined using the sensitive TRAP (telomerase rapid amplification protocol) assay in 45 nasopharyngeal biopsies (36 NPC, nine normal nasopharyngeal mucosae) in four xenografted NPC tumours established in nude mice and in five in vitro NPC cell lines. Telomerase activation is common in NPC and can be detected at high frequencies (85% in primary tumours and 100% in recurrent tumours). The frequency of telomerase activation was lowest in NPC biopsies without lymph node involvement (60%) compared with those with positive lymph node involvement (100%), and the difference is statistically significant (P < 0.05; Fisher exact test). All the xenografted NPC tumours and in vitro NPC cell lines were strongly positive for telomerase activity. Our results suggest that telomerase activation is common in NPC and it may be useful as a diagnostic marker in the detection of tumour cells in nasopharyngeal biopsies. The high frequency of telomerase activation in stage I NPC (80% positive) suggests that it is an early event in tumour progression.     

hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells

Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited telomerase activity but had no effect on the expressions of telomerase core subunits. RNA interference (RNAi) and in vitro phosphorylation studies revealed that PKC isoforms alpha, beta, delta, epsilon, zeta specifically involved in telomerase regulation, and the phosphorylation target was on hTERT. Treatment with the hsp-90 inhibitor novobiocin dissociated hsp90 and hTERT as revealed by immunoprecipitation and immunoblot analysis and reduced telomerase activity. Treatment with the PKC activator SC-10 restored the association of hsp90 and hTERT and reactivate telomerase, suggesting that hTERT phosphorylation by PKC is essential for telomerase holoenzyme integrity and function. Analysis on clinical normal and tumour tissues reveal that the expressions of PKC alpha, beta, delta, epsilon, zeta were higher in the tumour tissues, correlated with telomerase activity. Disruption of PKC phosphorylation by BIS significantly increased chemosensitivity to cisplatin. In conclusion, PKC isoenzymes alpha, beta, delta, epsilon, zeta regulate telomerase activity in head and neck cancer cells by phosphorylating hTERT. This phosphorylation is essential for telomerase holoenzyme assembly, leading to telomerase activation and oncogenesis. Manipulation of telomerase activity by PKC inhibitors is worth exploring as an adjuvant therapeutic approach.     

Effect of environmental conditions (pH, oxygenation and temperature) on the cytotoxicity of flavone acetic acid and its dimethylaminoethyl ester.

Bioflavonoids are known to inhibit enzymes in the glycolytic pathway and have been reported to decrease tumour blood flow. The antineoplastic capabilities of flavone acetic acid (FAA), dimethylaminoethyl-flavone-8-acetate (FAA ester) and quercitin (Q) as a function of pH, level of oxygenation and in conjunction with hyperthermia or SR-4233. In vitro, exposure of FSaIIC murine fibrosarcoma cells to various concentrations of FAA or FAA ester for 1 h demonstrated that both drugs were slightly more toxic toward hypoxic cells at 37 degrees C and pH 7.40 (but were somewhat less cytotoxic at pH 6.45 and 37 degrees C) than towards normally oxygenated cells. The cytotoxicity of FAA and FAA ester increased only minimally by concomitant treatment of cells at 42 degrees C or 43 degrees C. When temperatures of tumour-bearing mice anaesthetized with chloral hydrate and pentobarbital were measured both FAA (200 mg/kg) and Q (200 mg/kg) caused a more rapid drop in tumour versus core temperature, indicating a relative shutdown of tumour blood flow had been produced by these flavonoids. In Hoechst 33342 dye-defined subpopulations, both FAA and Q were only minimally cytotoxic in the subpopulation enriched in euoxic (bright) cells, producing surviving fractions of 0.70 and 0.29, respectively but were approximately 2-fold and 3-fold respectively more toxic towards the subpopulation enriched in hypoxic (dim) cells. When FAA preceded hyperthermia approximately a 3-4-fold increase in cell kill resulted from the combination in both subpopulations. Finally, when SR-4233, a selective hypoxic cell cytotoxic agent, was administered prior to FAA or Q and followed by hyperthermia the level of tumour cell killing increased so that the surviving fractions were 0.009 and 0.0055, respectively, in the dim cell subpopulation. These results indicate that FAA, FAA ester and Q may be most effectively used in a setting involving a combined modality regimen with a focus on the hypoxic tumour cell population.     

Clinical significance of telomerase activation and telomeric restriction fragment (TRF) in cervical cancer

Telomerase activation was examined in 50 cases of cervical cancer, 27 normal cervix and five cervical cancer cell lines using the sensitive polymerase chain reaction (PCR)-based TRAP (telomeric repeat amplification protocol) assay. Telomeric restriction fragment (TRF) length of these specimens was measured by Southern hybridisation. Telomerase activation was common in cervical cancers and was detected in 46/50 cases (92%). Telomerase activity was weak in normal cervix and was detected only in 2/27 cases (7.4%). Telomerase activity was detected in all stages of cervical cancer suggesting that it is an early event in cancer progression. The clinical significance of telomerase activation was analysed in 47 squamous cell carcinoma of the cervix. High telomerase activity was more frequently detected in advanced diseases (100% in stage III and stage IV cervical cancers combined) compared with early diseases (68.6% in stage I and stage II cancers combined). The difference was statistically significant (P < 0.02). Telomerase activity was not statistically correlated with other clinical parameters examined. This is the first report of telomeric length in human cervical cancer. Both shortening and elongation of TRF length in cervical cancers was observed. Advanced cervical cancers tended to have a wider range of variation of TRF length compared with early disease and normal cervix. There was no obvious relationship between TRF length and the clinical parameters examined including clinical staging, differentiation status of tumour, human papilloma virus (HPV) infection, recurrence rate, tumour size and invasion depth. The clinical significance of TRF length appears to be limited in cervical cancers. Our results indicate that telomerase activity is closely associated with tumour cells and may be useful as a marker for detection of tumour cells in cervical biopsies.     

Apoptosis induced by hyperthermia in Dunn osteosarcoma cell line in vitro

The effect of hyperthermia at 43.5^oC for 1h on Dunn osteosarcoma cells was studied. With sham-heated cells (37^oC, 1h) as the control, the hyperthermia treated cells were divided into five groups. Time 0 group was the cells that were harvested immediately after heated at 43.5^oC for 1h. Whereas time 3, 6, 12, and 24h groups were the cells that were collected respectively after reincubation at 37^oC for the above different time periods. The appearance of hyperthermiainduced apoptosis of Dunn osteosarcoma cells was demonstrated to be time dependent. With the confocal microscopic study and TUNEL staining, the morphological characteristics of apoptosis, condensed nuclei and fragmented nuclei were obvious when reincubated at 37^oC for 6h after hyperthermic treatment. This hyperthermia-induced apoptosis was further confirmed by flow cytometric analysis on DNA contents. The sub-G₁ region that was proposed as a marker of apoptotic cells was most significantly elevated at 6h after hyperthermic treatment and, thereafter, decreased to the levels of control values by 24h, as the apoptotic cells underwent secondary necrosis and degraded to debris. The DNA strand breaks, considered as the key biochemical event of apoptosis, were detected by the TUNEL assay. This study indicated that hyperthermia (43.5^oC for 1h) can induce apoptotic changes on osteosarcoma cells in vitro very rapidly (within 6h after treatment), and its occurrence might not be detected if the samples are not taken at several early time points after hyperthermia.     

Effects of hyperthermia on natural killer cells: inhibition of lytic function and microtubule organization.

Cells with natural killer activity (NK) may play an important role in host defence against tumour cells. The lytic function of NK cells is very sensitive to hyperthermic inactivation. However, cells with NK activity isolated from rat spleen and exposed to 41-42.5 degrees C for 30 min could partially recover their cytotoxic activity after incubation at 37 degrees C. The recovered cytotoxicity was still NK-specific, as it only resulted in the lysis of YAC-1 sensitive targets, and could not lyse NK-resistant P815 mastocytoma cells. Conjugate formation assay using NK cells labelled with specific monoclonal antibody (mAb) 3.2.3 indicated that the binding of NK cells to targets was not significantly affected by heat treatment. Compared to controls, however, microtubule organizing centre (MTOC) reorientation towards the region of intercellular contact was reduced by 40% in heated effector cells. This was accompanied by a greater inhibition (62-77%) of NK lytic activity. Kinetic analysis indicated that MTOC reorientation capacity recovered following incubation at 37 degrees C. MTOC recovery was maximal 4 h after treatment whereas that of lytic activity peaked at 6 h. These data indicate that NK cells recover NK-specific lytic activity after heat inactivation. Moreover, our study demonstrates that hyperthermia interferes with post-binding MTOC reorientation, and further supports a role for microtubule in secretory processes involved in NK-mediated cytolysis.     

Heat induced release of Hsp70 from prostate carcinoma cells involves both active secretion and passive release from necrotic cells.

PURPOSE: Heat shock protein 70 (HSP70) is released from tumour cells and stimulates a potent anti-tumour immune response. METHODS: This study examined the role of hyperthermia, including heating conditions from the fever range, the hyperthermia range and the thermal ablation range, in HSP70 release from prostate carcinoma cells. It has observed HSP70 release from human prostate carcinoma cell lines (PC-3 and LNCaP) treated with hyperthermia. RESULTS: The effects of hyperthermia were complex and appeared to involve at least two mechanisms for HSP70 release. Hyperthermia at 40 degrees C strongly stimulated HSP70 release by an active secretion pathway. However, as temperatures were increased, this rapid secretion pathway became progressively inhibited and by a temperature of 55 degrees C, active secretion was abolished. However, when cells exposed to these heating conditions were allowed to recover at 37 degrees C for 24 h after heating, HSP70 release was observed at the high ablation temperature range and this appeared to be related to a concomitant damage to the plasma membrane. CONCLUSIONS: Thus, at least two mechanisms contribute to HSP70 release during hyperthermia and the relative contribution from each pathway depends on the temperature conditions.     

Effect of 41 degrees C and 43 degrees C on cisplatin radiosensitization in two human carcinoma cell lines with different sensitivities for cisplatin

The effect of trimodality treatment consisting of hyperthermia, cisplatin and radiation was investigated in two cell lines with different sensitivities to cisplatin. Hyperthermia treatment was performed for 1 h at 41 degrees C and 43 degrees C in order to compare the effects of the two temperatures. Clonogenic assays were performed with cisplatin-sensitive SiHa human cervical carcinoma and cisplatin-resistant SW-1573 human lung carcinoma cell lines. Cells were treated with various combinations of hyperthermia, cisplatin and radiation. Radiation was performed after 1 h of simultaneous hyperthermia and cisplatin treatment. Cisplatin exposure was for 1 h or continuous without refreshment of the cisplatin-containing medium. SiHa cells were more sensitive to cisplatin than SW-1573 cells. Hyperthermia at 41 degrees C decreased survival in SW-1573 cells but was not cytotoxic in SiHa cells. Hyperthermia at 43 degrees C decreased survival dramatically in both cell lines with SiHa being the most sensitive. The addition of hyperthermia at 41 degrees C and 43 degrees C to cisplatin treatment led to enhanced cell kill in both cell lines compared with cisplatin alone. Radiosensitization was observed after continuous but not after 1 h of cisplatin treatment. Hyperthermia at 43 degrees C increased radiosensitivity whereas hyperthermia at 41 degrees C did not. A combination of 41 degrees C hyperthermia with continuous cisplatin treatment had an additive effect on SW-1573 cells but enhanced cisplatin radiosensitivity of SiHa cells. In SW-1573 cells trimodality treatment using 43 degrees C hyperthermia enhances cisplatin radiosensitivity. We conclude that hyperthermia at 43 degrees C enhances cisplatin-induced radiosensitization in both cisplatin-sensitive and -resistant cell lines. Hyperthermia at 41 degrees C was also able to increase cisplatin-induced radiosensitivity but only in the cisplatin-sensitive SiHa cell line.     

Poly (ADP-ribose) polymerase 3 (PARP3), a potential repressor of telomerase activity

Background

Considering previous result in Non-Small Cell Lung Cancer (NSCLC), we investigated in human cancer cells the role of PARP3 in the regulation of telomerase activity.

Methods

We selected A549 (lung adenocarcinoma cell line) and Saos-2 (osteosarcoma cell line), with high and low telomerase activity levels, respectively. The first one was transfected using a plasmid construction containing a PARP3 sequence, whereas the Saos-2 cells were submitted to shRNA transfection to get PARP3 depletion. PARP3 expression on both cell systems was evaluated by real-time quantitative PCR and PARP3 protein levels, by Western-blot. Telomerase activity was determined by TRAP assay.

Results

In A549 cells, after PARP3 transient transfection, data obtained indicated that twenty-four hours after transfection, up to 100-fold increased gene expression levels were found in the transfected cells with pcDNA/GW-53/PARP3 in comparison to transfected cells with the empty vector. Moreover, 48 hours post-transfection, telomerase activity decreased around 33%, and around 27%, 96 hours post-transfection. Telomerase activity average ratio was 0.67 ± 0.05, and 0.73 ± 0.06, respectively, with significant differences. In Saos-2 cells, after shRNA-mediated PARP3 silencing, a 2.3-fold increase in telomerase activity was detected in relation to the control.

Conclusion

Our data indicated that, at least in some cancer cells, repression of PARP3 could be responsible for an increased telomerase activity, this fact contributing to telomere maintenance and, therefore, avoiding genome instability.     

恶性淋巴瘤中端粒酶活性及其意义

目的：研究淋巴瘤细胞株及淋巴瘤组织中的端粒酶活性及其在淋巴瘤发生过程中的作用。方法：用端粒重复序列扩增法（ＴＲＡＰ）结合银染,检测３株人淋巴瘤细胞株、２７例淋巴瘤和４例淋巴结良性病变的端粒酶活性。结果：３株人淋巴瘤细胞株均有端粒酶活性,其中２例活性较弱,低于淋巴瘤。结论：恶性淋巴瘤中存在端粒酶的活性表达,但有必要采用定位研究来识别具有端粒酶活性的细胞,对端粒酶阳性表达在淋巴组织疾患中的解释需特别慎     

Telomerase in relation to expression of p53, c-Myc and estrogen receptor in ovarian tumours

Telomerase activity and its subunits (hTERC, hTERT mRNA) were evaluated in ovarian tumours in relation to the expression of p53, c-Myc and estrogen receptor (ER). Furthermore, relations between telomerase activity, hTERC and hTERT with known clinicopathologic prognostic factors and survival in patients with malignant tumours was investigated. Telomerase activity was determined with TRAP, hTERC and hTERT with RT-PCR, while p53, c-Myc and ER expression with immunohistochemistry. Telomerase activity and hTERT mRNA were more frequently observed in malignant ovarian tumours compared to borderline and benign tumours, whereas hTERC was present in all tumour types. p53 and c-Myc were more frequently detected in malignant compared to borderline and benign tumours. Telomerase activity was positively related to hTERT mRNA, p53 and c-Myc expression, but not to hTERC and ER expression. In malignant tumours, hTERC levels were related to tumour stage, while telomerase activity and hTERT mRNA expression were not related to any clinicopathologic feature. Tumour stage, differentiation grade, residual tumour after first laparotomy and presence of ascites were related to (progression free) survival, whereas telomerase activity or its subunits were not. In conclusion, these data suggest that p53 expression (e.g. p53 mutation) as well as c-Myc expression may have a role in regulation of telomerase activity in ovarian tumours.     

Simultaneous targeting of telomeres and telomerase as a cancer therapeutic approach

Telomeres, which are important for maintaining chromosome integrity and functions, shorten with each cell division. Telomerase, responsible for telomere synthesis, is expressed in approximately 90% of human tumor cells but seldom in normal somatic cells. This study evaluated the hypothesis that simultaneous shortening of telomeres and inhibition of telomerase results in synergistic and tumor-selective cytotoxicity. In telomerase-positive human pharynx FaDu tumor cells, paclitaxel caused telomere erosion (first detected at 1 h) and apoptosis. Expression of antisense to the RNA component of human telomerase (hTR) inhibited telomerase activity, shortened telomere length, reduced cell growth rate, and resulted in a significant higher sensitivity to paclitaxel. Another telomerase inhibitor, 3'-azido-3'-deoxythymidine (AZT), at a concentration that produced little or no cell detachment or apoptosis, inhibited the telomerase activity and enhanced the paclitaxel-induced cell detachment and apoptosis. AZT also enhanced the activity of paclitaxel in mice bearing well-established s.c. FaDu xenograft tumors (i.e., reduced residual tumor size, enhanced apoptotic cell fraction, and prolonged survival time), without enhancing host toxicity. In contrast, AZT did not enhance the paclitaxel activity in the telomerase-negative osteosarcoma Saos-2 cells nor in FaDu cells where telomerase was already suppressed by antisense hTR, confirming that the AZT effect in parent FaDu cells is mediated through telomerase inhibition. These results demonstrate that combined use of agents targeting both telomere and telomerase yielded synergistic activity selective for tumors that depend on telomerase for telomere maintenance.     

Telomerase activity and Bcl-2 expression in human breast cancer.

AIMS: Telomerase is a ribonucleoprotein that synthesizes telomeres and plays an important role in cellular immortalization. Bcl-2 gene encodes for a mitochondrial protein thought to prevent apoptosis of normal cells. We previously reported telomerase activity in 74% of human invasive breast cancers and detected a significant association between telomerase activity and prognostic parameters such as nodal status, tumour size and cellular proliferation. We hypothesized that telomerase reactivation in human breast cancer was associated with increased immunohistochemical expression of Bcl-2. METHODS: Bcl-2 immunohistochemical expression was determined in 25 infiltrating breast carcinomas with known telomerase activity (17 telomerase-positive and 8 telomerase-negative). The percentage of strongly and moderately stained tumour cells for Bcl-2 was determined by a breast pathologist who was blinded to telomerase data. Fisher's exact test was used to examine the association between telomerase activity and Bcl-2 expression. RESULTS: The median percentage of strongly stained tumour cells was 50% for telomerase-positive tumours (range, 0--100%) and 45% for telomerase-negative tumours (range, 0--100%). Twelve (70%) of 17 telomerase-positive tumours expressed strong or moderate Bcl-2 staining in >50% of tumour cells compared with six (75%) of eight telomerase-negative tumours (P=1.0). CONCLUSION: Telomerase reactivation seems to be independent of Bcl-2 protein expression in human breast cancer.     

Induction of thermotolerance and heat-shock protein synthesis during nutritional deprivation.

Under various conditions of heating, H35 cells were submitted to acute nutritional deprivation by omitting a number of substrates (L15D medium). At 37 degrees C cell death starts after a lag-period of 3-5 h. During hypothermia cell death is delayed, whereas during hyperthermia it is accelerated especially as a result of thermosensitization. In L15D the ATP level decreases approximately 3 times faster in combination with hyperthermia than at 37 degrees C. In non-thermotolerant cells thermosensization is very high at 41 degrees C and decreases with increasing temperature; in thermotolerant cells it is comparatively decreased at 41 degrees C and increased at 42.5 degrees C and above. In response to a heat shock of 30 min at 42.5 degrees C only 10% of the cell population expresses acute thermotolerance after incubation at 37 degrees C in L15D as compared to nearly 100% in complete medium (L15C). Chronic development of thermotolerance appears to be even more repressed in the presence of L15D, which partly explains the high thermosensitization at 41 degrees C. Changes in the rate of protein synthesis for combinations of nutritional deprivation and hyperthermia show a correlation with the cell survival data. Development of acute thermotolerance in L15D is accompanied by an increase in heat-shock protein synthesis relative to total protein. At 41 degrees C in L15D no heat-shock protein induction could be detected. Of the omitted substrates only glutamine can effectively abolish thermosensitization and the effects of L15D on protein and heat-shock protein synthesis depending on the condition of the cells, thermotolerant or non-thermotolerant, and to a different extent for the various proteins considered.