Correlating telomerase activity levels with human neuroblastoma outcomes.

Telomerase activity was analysed in 100 neuroblastoma cases. Although telomerase activity was not detected in normal adrenal tissues or benign ganglioneuromas, almost all neuroblastomas (94%) did express it, suggesting an important role for telomerase in neuroblastoma development. Neuroblastomas with high telomerase activity had other genetic changes (for example, N-myc amplification) and an unfavourable prognosis, whereas tumours with low telomerase activity were devoid of such genetic alterations and were associated with a favourable prognosis. Three neuroblastomas lacking telomerase activity regressed (stage IVS). Thus telomerase expression may be required as a critical step in the multigenetic process of tumorigenesis, and two different pathways may exist for the development of neuroblastoma.

Synthesis of an azadioxa-planar triphenylborane and investigation of its structural and photophysical properties.

We report here the first successful synthesis of planar triphenylborane 1 with the phenyl groups bridged by oxygen and nitrogen atoms via double nucleophilic aromatic substitution reaction. The hetero atom-bridged 1 has excellent planarity. Its structural and photophysical properties are tunable by altering the bridging atoms.

Telomerase activity in human breast tumors.

BACKGROUND: The activity of the ribonucleoprotein enzyme telomerase is not detected in normal somatic cells; thus, with each cell division, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode. The current model gaining support is that telomerase activity in germline and immortal cells maintains telomere length and thus compensates for the "end-replication problem." PURPOSE: Our objective was to determine when telomerase activity is reactivated in the progression to malignant breast cancer and if knowledge of telomerase activity may be an indicator for the diagnosis and potential treatment of breast cancer. METHODS: Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 140 breast cancer specimens (from 140 patients), four phyllodes tumors (from four patients), 38 noncancerous lesions (20 fibroadenomas, 17 fibrocystic diseases, one gynecomastia; from 38 patients), and 55 adjacent noncancerous mammary tissues (from 55 of the 140 breast cancer patients). In addition, 33 fine-needle-aspirated breast samples (from 33 patients) were analyzed. RESULTS: Among surgically resected samples, telomerase activity was detected in 130 (93%) of 140 breast cancers. Telomerase activity was detected in 68% of stage I primary breast cancers, in 73% of cancers smaller than 20 mm, and in 81% of axillary lymph node-negative cancers. Moreover, the activity was detected in more than 95% of advanced stage tumors but in only two (4%) of 55 adjacent noncancerous tissues. While telomerase activity was not detected in any of 17 specimens of fibrocystic disease, surprisingly low levels of telomerase activity were detected in nine (45%) of 20 fibroadenomas. Among samples obtained by fine-needle aspiration, 14 (100%) of 14 patients whose fine-needle-aspirated specimen contained telomerase activity and who subsequently underwent surgery were confirmed to have breast cancer. Multivariate analysis of 125 specimens from patients for whom data were available on age at surgery, stage of disease, tumor size, lymph node status tumor histology, and menopausal status indicated that stage classification exhibited the strongest association with telomerase activity (for stage I versus stages II-IV: odds ratio = 1.0 versus 73.4; 95% confidence interval = 2.0-959.0; P = .02). CONCLUSION: Telomerase activity was detected in more than 95% of advanced stage breast cancers. It was absent in 19%-32% of less advanced cancers. Since a determination of any association between telomerase activity and patient survival is not possible at the present time, it remains to be determined whether lack of telomerase activity predicts for favorable outcome.

Telomerase activity in small-cell and non-small-cell lung cancers.

BACKGROUND: Telomerase is an enzyme that adds hexameric TTAGGG nucleotide repeats onto the ends of vertebrate chromosomal DNAs (i.e., telomeres) to compensate for losses that occur with each round of DNA replication. Somatic cells do not have telomerase activity and stop dividing when the telomeric ends of at least some chromosomes have been shortened to a critical length. It has been suggested that immortalized cells (including some, but probably not all, cancer cells) continue to proliferate indefinitely because they express telomerase. PURPOSE: To investigate whether expression of telomerase is a prerequisite for the development of naturally occurring human cancers, we assayed the levels of telomerase activity in specimens of human lung tumor and adjacent normal tissue. METHODS: Using a polymerase chain reaction-based assay, we examined telomerase activity in 136 primary lung cancer tissues and 68 adjacent noncancerous tissues obtained by surgical resection. We also studied telomerase activity in four primary and 23 metastatic lesions obtained through biopsy, (two patients) or autopsy (10 patients). Relative telomerase activity levels were estimated by serial dilutions of extracts prepared from the specimens. Telomerase activity was also assayed in extracts of cells present in pleural fluids from three patients with adenocarcinoma of the lung. RESULTS: Among surgically resected samples, telomerase activity was detected in 109 (80.1%) of 136 primary lung cancer tissues and in three (4.4%) of 68 normal adjacent tissues. All 11 surgically resected specimens of primary small-cell lung cancer (from 11 patients) revealed high levels of telomerase activity, whereas the activity ranged from undetectable to high levels in the 125 surgically resected specimens of primary non-small-cell lung cancer tissue (from 125 patients). Generally, high levels of telomerase activity were observed in metastatic lesions and tumors with altered telomere length. A few primary and, surprisingly, some metastatic tumors did not appear to have detectable telomerase activity. Telomerase activity was, however, detected in cells present in all tested pleural fluids obtained (from three patients with adenocarcinoma of the lung). CONCLUSION: The subset of non-small-cell lung cancers that exhibits only low or undetectable levels of telomerase activity may contain primarily mortal cancer cells. Cancers that exhibit high levels of telomerase activity, such as all of the small-cell lung cancers examined in this study, are likely to consist mainly of immortal cells. IMPLICATIONS: Telomerase activity may be useful both as a diagnostic marker to detect the existence of immortal lung cancer cells in clinical materials and as a target for therapeutic intervention.

Neuroblastoma with DNA amplification and rearrangement in the N-myc gene region.

We detected a rearrangement in the N-myc gene region in a neuroblastoma from a 9-month-old girl. In this case, the N-myc gene was amplified 50-fold in the primary tumor, the lymph node metastasis, and the hepatic metastasis. The rearrangement was detected only in the primary tumor and the lymph node metastasis, whereas N-myc RNA and protein expression were detected only in the primary tumor and the hepatic metastasis. Both the rearranged N-myc gene and the normal N-myc gene were amplified 25-fold, and the rearrangement occurred 723 nucleotides downstream from the 3' end of exon 3. The cell line (NH-6) derived from the primary tumor showed N-myc gene amplification without this rearrangement. These results suggest the following: (a) the primary tumor had a least two clones; (b) the rearrangement interrupted N-myc gene expression; and (c) oncogenesis preceded N-myc gene amplification.

Telomerase activity in gastric cancer.

Although many genetic alterations have been reported in gastric cancer, it is not known whether all gastric tumors are capable of indefinite proliferative potential, e.g., immortality. The expression of telomerase and stabilization of telomeres are concomitant with the attainment of immortality in tumor cells; thus, the measurement of telomerase activity in clinically obtained tumor samples may provide important information useful both as a diagnostic marker to detect immortal cancer cells in clinical materials and as a prognostic indicator of patient outcome. Telomerase activity was analyzed in 66 primary gastric cancers with the use of a PCR-based assay. The majority of tumors (85%) displayed telomerase activity, but telomerase was undetectable in 10 tumors (15%), 8 of which were early stage tumors. Most of the tumors with telomerase activity were large and of advanced stages, including metastases. Survival rate of patients of tumors with detectable telomerase activity was significantly shorter than that of those without telomerase activity. Alterations of telomere length (reduced/elongated terminal restriction fragments) were detected in 14 of 66 (21%) gastric cancers, and all 14 had telomerase activity. Cellular DNA contents revealed that all 22 aneuploid tumors had detectable telomerase activity. The present results indicate that telomerase activation may be required as a critical step in the multigenetic process of tumorigenesis, and that telomerase is frequently but not always activated as a late event in gastric cancer progression.

Telomerase activity is detected in pancreatic cancer but not in benign tumors.

Activation of telomerase and stabilization of telomeres are considered to be necessary for immortalization of human tumor cells. In the present study, telomerase activity was detected in 41 (95%) of 43 pancreatic cancer specimens but was detectable in none of 11 benign pancreatic tumors and only one of 3 pancreatitis samples. Low levels of telomerase activity were detected in 5 (14%) of 36 adjacent "normal" pancreatic tissues. These five telomerase-positive "normal" specimens were obtained from patients that also had pancreatic cancer and may reflect occult microinvasion. Telomerase activity was examined in 12 ex vivo brushing samples of the pancreatic duct, and 8 of 8 with pancreatic cancer had detectable telomerase activity, whereas 0 of 4 of benign lesions (cystadenoma and pancreatitis) did. These findings suggest that telomerase activity in cells derived from pancreatic ducts may be useful in the diagnosis of cancer and that telomerase activity may be a critical or rate-limiting step in pancreatic carcinogenesis.

Alterations in telomeric repeat length in lung cancer are associated with loss of heterozygosity in p53 and Rb.

In the two-stage model of controlling cellular senescence in cultured human fibroblasts, retinoblastoma (Rb) and p53 proteins may be key factors regulating the mortality stage 1 mechanism. In addition, the critical loss of telomeric DNA due to the end-replication problem may result in the mortality stage 2 mechanism. Cells which acquire telomerase activity can overcome the M2 mechanism by stabilizing telomere length and thus become immortal (telomere hypothesis). At present it is known whether cellular immortality is a prerequisite for all human cancers. To investigate this question and the applicability of the two-stage model to human cancers, we analysed the relationship between alterations of telomere length and other genetic changes in lung cancer. Among 60 primary lung cancer tissues, telomere length alterations were observed in 16 tumors (26.7%) including 14 with short and two with elongated telomeres. Ten of them revealed allelic loss of both p53 and Rb genes, and remaining six showed no abnormalities in both genes. We propose that inactivation of both p53 and Rb genes may promote cell divisions causing telomere shortening in lung cancer as in the two-stage model, while there may be another pathway to overcome both M1 and M2 mechanisms, especially for adenocarcinoma.

p53 gene mutations are associated with shortened survival in patients with advanced non-small cell lung cancer: an analysis of medically managed patients.

Mutations in the p53 gene are common in many cancers. Nevertheless, the relationship between mutations of this tumor suppressor gene and patient survival in non-small cell lung cancer (NSCLC) remains unclear. Interpretation of prior studies of patient outcomes are complicated by the inclusion of both surgical and nonsurgical patients. To better isolate the potential effects of p53 gene mutations per se on tumor progression, we chose to examine patients with advanced disease in whom surgery was not performed (stages IIIA, IIIB, and IV). We have used PCR-denaturing gradient gel electrophoresis, a sensitive and specific method for the detection of a variety of p53 mutations in cytology or biopsy specimens, to evaluate the prognostic significance of p53 gene mutations in nonsurgical patients with advanced NSCLC. In 70 consecutive medical patients, p53 mutations were found in 29 cases (41%) at the time of initial diagnosis. Followed prospectively, patients with p53 mutations had a significantly reduced survival time after diagnosis than those without mutations (median survival, 17 versus 39 weeks; P = 0.0003) independent of other clinical factors. This abbreviated survival occurred in both patients who received chemotherapy (n = 39, P = 0.002) or best supportive care (n = 31, P = 0.018). These results indicate that mutations of the p53 gene in patients with NSCLC who do not undergo surgical resection portends a significantly worse prognosis.

Rapid detection of MYCN gene amplification and telomerase expression in neuroblastoma.

Amplification of the MYCN gene and high telomerase activity predict a poor prognosis for the patients with neuroblastoma. We used PCR techniques for rapid detection of MYCN gene amplification and human telomerase reverse transcriptase (hTERT) expression in neuroblastoma specimens. The detection of MYCN gene amplification is based on differential PCR in which three primer pairs were used to coamplify a 178-bp fragment of target MYCN gene with two reference gene fragments, a 237-bp of p53 exon 7 and a 120-bp of beta-globin exon 3, in a single tube of 40 surgically resected tumor samples. MYCN amplification was identified by this differential PCR in all 10 samples carrying more than 10 copies (already known to have MYCN gene amplification by Southern blot analysis). There were no false-negative or false-positive cases, and the relative intensity of MYCN bands in the differential PCR correlated significantly with the copy number determined by Southern blot analysis (y = 0.99, P<0.0001). This protocol was also applicable in the biopsy or aspirated samples, as well as the paraffin-embedded tissues, and in detecting intratumoral heterogeneity. Using RT-PCR procedures, hTERT mRNA expression was detectable in all 13 tumors with high telomerase activity. These nonradioisotopic PCR-based protocols for detecting MYCN gene amplification and hTERT mRNA expression are rapid and reliable and are likely to be useful to determine the biological behavior of neuroblastoma.

Immunohistochemical detection of telomerase (hTERT) protein in human cancer tissues and a subset of cells in normal tissues.

We examined human telomerase reverse transcriptase (hTERT) protein distribution by immunohistochemistry in cultured cells and tissue sections. Cells with telomerase activity had nuclear positive signals whereas cells without telomerase activity did not. In most normal epithelial tissues, hTERT expression was prominent in the early proliferative descendent progenitors cells. In cancers with high telomerase activity, hTERT expression was detected in almost all neoplastic cells and correlated with telomerase activity levels, whereas cancers with low telomerase activity had fewer hTERT-positive cancer cells. In pediatric neuroblastomas with a favorable outcome, both the percentage of positive cells and the signal intensities of each hTERT-expressing cell decreased. These studies indicate that detection of telomerase at the cellular level is achievable and may have utility in cancer diagnostics.

Allelotype frequency of the thiopurine methyltransferase (TPMT) gene in Japanese.

Polymorphisms at three loci in the thiopurine methyltransferase (TPMT) gene are known to be responsible for azathioprine and 6-mercaptopurine (6MP) toxicity. Among them, only TPMT*3C variant allele with A719G mutation was found in 15/522 (2.9%; 17/1044 alleles; 1.6%) Japanese individuals including two homozygotes. The allele frequency was different from that in Caucasians, and investigation of TPMT polymorphisms with consideration of ethnic differences before administration of azathioprine or 6MP may provide clinically useful information.

Telomerase activity in neuroblastoma: is it a prognostic indicator of clinical behaviour?

Neuroblastomas show remarkable biological heterogeneity, resulting in favourable prognosis or unfavourable prognosis due to aggressive growth despite multimodal therapy. Recently, we proposed that aggressive tumours express telomerase at a high level while the favourable tumours lack or have low telomerase expression. To evaluate the correlation between telomerase activity and other biological characteristics reported as prognostic markers (MYCN gene amplification, loss of heterogeneity (LOH) in the short arm of chromosome 1, trk-A expression, Ha-ras p21 expression, and DNA ploidy), we investigated these biological features in 105 untreated neuroblastomas. In these cases, 23 showed high telomerase activity, 78 showed low activity, and telomerase activity was undetectable in 4 cases. Most tumours with genetic alterations (MYCN amplification or 1p32 LOH) showed high telomerase activity. Most tumours with low or undetectable activity were aneuploid, and showed trk-A and Ha-ras expression. Three of the four tumours with undetectable telomerase activity regressed. In 2 of the tumours with low telomerase activity, the residual tumours maturated and showed repression of telomerase activity. Thus, the level of telomerase activity correlated with other genetic alterations and/or gene expression and may be a useful prognostic indicator in neuroblastoma.

Former poison gas workers and cancer: incidence and inhibition of tumor formation by treatment with biological response modifier N-CWS.

Mustard gas is known to have mutagenic and carcinogenic effects on animal and human cells. In this report, 1,632 male Japanese who worked in poison gas factories at some time between the years 1927 and 1945 were studied to determine comparative risk for development of cancer, the reference population being data on Japanese males overall. The standardized mortality ratio (SMR) for lung cancer in workers directly and indirectly involved in the production of mustard gas was significantly elevated. In addition, SMR for lung cancer in worker who had worked for more than 5 years was also significantly elevated. Thus, poison gas workers who had engaged in the production of mustard gas or related work for more than 5 years are a high-risk group for lung cancer. Under the cancer preventive program, Nocardia rubra cell-wall skeleton (N-CWS) was administered to 146 former poison gas workers. During a 4.5 year observation period, development of cancers was found in 7 treated workers and 17 untreated controls. After elimination of the influence of smoking level, a significant suppression of development of cancers was noted in the N-CWS-treated workers as compared to the untreated controls. Although the molecular mechanisms of carcinogenesis in former poison gas workers remains unclear, our study proposes the possible effect of biological response modifiers in the prevention of cancer development in high-risk human subjects.

Alternative splicing, but not allelic loss, of the FHIT gene increases with development of lung cancer.

The FHIT gene is considered to be a tumor suppressor gene, its role and inactivation mechanism remain unclear. We analyzed FHIT gene aberrations in 64 lung cancer tissues and found that the appearance of the aberrant FHIT transcripts depends on the condition of RT-PCR and high telomerase activity, shortened telomere length, and advanced pathological stage were likely associated with the prevalence of aberrant FHIT transcripts, but not with allelic loss of the FHIT gene. These observations would indicate that an additional unknown gene may exist, which is more responsible for the allelic loss around the FHIT gene locus.

Alteration of telomeric repeat length in adult and childhood solid neoplasias.

The ends of human chromosome contain unique structures referred to as telomeres which confer genetic stability to the chromosomes, but due to the end replication problem, cell division both in vivo and in vitro reduce the average telomere length in normal somatic cells. We have measured the length of terminal restriction fragments (TRFs) at the telomeric ends in 215 adult and 156 pediatric solid neoplasias. Reduced TRFs were detected in 52 adult tumors (24.2%) and 33 pediatric tumors (21.2%), while elongated TRFs were detected in 19 adult tumors and seven pediatric tumors. Our results indicate that while TRF lengths are altered in a fraction of various kinds of tumor, many tumors consist of cells with TRF lengths which do not differ from those of adjacent normal tissue.

Telomerase activity in human intestine.

In human somatic cells without the activity of telomerase, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode with each cell division. In germline and immortal cells telomerase activity maintains telomere length and thus compensates for the 'end-replication problem'. Progressive telomere shortening and reactivation of telomerase activity have been considered to be one of the key mechanisms in cellular senescence and immortalization. It has been shown that while most somatic cells do not have detectable telomerase activity, almost all cancers do have telomerase activity. Thus, detection of telomerase activity may have utility in the early diagnosis of cancer and may be a new target for therapeutic intervention. However, there is recent evidence that some cells of renewal tissues, such as hematopoietic cells and basal cells of the epidermis, have detectable telomerase activity. In the present study, we report detectable telomerase activity in normal human intestinal mucosa. This activity is localized to the lower third of each crypt and may be derived from intestinal stem cells. Since intestinal telomeric repeats are shorter in adults when compared to children, the telomerase activity in the intestine is insufficient to maintain telomere length but may be sufficient to provide extended proliferative capacity for such renewal tissues.

An atypical carcinoid tumor of the lung with mutations in the p53 gene and the retinoblastoma gene.

Molecular analysis of a metastatic lesion of an atypical carcinoid tumor of the lung obtained from a 77-year-old man at autopsy revealed a point mutation in the p53 gene and a deletion in the retinoblastoma (Rb) mRNA. This case suggests that both these antioncogenes may be involved in the progression of atypical carcinoid tumor.

Increased expression of platelet-derived growth factor A and insulin-like growth factor-I in BAL cells during the development of bleomycin-induced pulmonary fibrosis in mice.

Current concepts suggest that macrophages may play a central role in pulmonary fibrosis by virtue of their ability to release a variety of cytokines. In this study, the expression of interleukin (IL)-1 alpha and beta, platelet-derived growth factor (PDGF) A and B, and insulin-like growth factor (IGF) I in BAL cells, which may be involved in fibroblast proliferation, was investigated in murine bleomycin (BLM)-induced pulmonary fibrosis. BAL cells were obtained at 1, 15, and 29 days from Institute for Cancer Research mice after 10 days of intraperitoneal administration of BLM. The relative amounts of cytokine messenger RNA (mRNA) were evaluated by the reverse transcription-polymerase chain reaction method, which simultaneously amplified complementary DNA for cytokines and beta-actin as an internal control. The level of IL-1 beta mRNA in BLM-treated mice was increased 4.5-fold compared with that in saline solution-treated (control) mice 1 day after treatment, while no significant differences were observed between the two groups at 15 and 29 days. The mRNAs of PDGF-A and IGF-I in BLM-treated mice were sustained at levels eightfold and threefold to fourfold, respectively, those of controls over 4 weeks. No significant differences were noted in IL-1 alpha and PDGF-B expression between the two groups. We conclude that IL-1 beta released from macrophages may be important in the early phase of inflammatory responses and that PDGF-A and IGF-I may play important roles in the development of BLM-induced pulmonary fibrosis.

Alteration in length of telomeric repeats in lung cancer.

We investigated the relationship between telomere length and various characteristics of tumor cells in 46 lung cancer specimens (40 primary lesions and six metastatic lesions). Three variant patterns of telomere length were observed in 16 cases (34.8%): reduction in 13 cases, elongation in two cases, and convergence in one case. These variant patterns were frequently observed in small cell carcinomas, in metastatic lesions, and in cases which possessed the S-type allele of the L-myc gene. All three cases with telomere elongation or convergence were associated with a poor prognosis. This is compatible with the previous report suggesting that telomerase activity may be an indicator of immortality in vitro. In adenocarcinoma, telomere reduction or elongation was also observed in the early stages with a low percentage of cells in the S-phase, while in cases with other histologic types, these changes were observed only in late stage, in metastatic lesions, or in cancerous tissues with a high percentage of cells in the S-phase. Although the reduction of telomere length in these tissues may be a result of many cell divisions, it may represent another stage of carcinogenesis in early-stage adenocarcinoma.