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.

Monoclonal antibodies to human squamous cell carcinoma of the lung and their application to tumor diagnosis.

Three immunoglobulin G1 monoclonal antibodies, LuCa2, LuCa3, and LuCa4, were produced by fusing murine myeloma NS1 cells with splenocytes obtained from a BALB/c mouse immunized with SK-MES1 cells derived from human squamous cell carcinoma of the lung. These three monoclonal antibodies were shown to recognize different protein antigens on SK-MES1 cells by indirect immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. While the pattern of cell line distribution of antigens recognized by these antibodies was not tumor type specific, their reactivity with tissue and pleural effusion was much more informative than with cell lines. The presence of target antigens in vivo was analyzed by immunoperoxidase staining of frozen tissue sections and immunofluorescence staining of tumor cells in pleural effusions. LuCa2 antibody was reactive with lung squamous carcinoma and adenocarcinoma tumor tissues and pleural effusions, but only infrequently with those of small cell carcinoma. This antibody was also reactive with many tumor tissues from other organs as well as with various normal tissues, including alveoli and bronchus. LuCa3 and LuCa4 antibodies reacted with lung squamous carcinoma in tissues and pleural effusions, but not with lung adenocarcinoma nor with small cell carcinoma. These two antibodies reacted only weakly with normal squamous tissues of the esophagus, skin, and cervix uteri, but not with various other normal tissues. Moreover, LuCa3 had weak reactivity with squamous cell carcinoma tissue of tongue and esophagus, whereas LuCa4 had no reactivity with nonpulmonary tumor tissues. LuCa3 and LuCa4 antibodies should be of clinical interest, because our data suggest that these antibodies may be potentially useful for the diagnosis of the histological type of lung tumor cells in both cancer tissue and pleural effusions.

Detection of a circulating tumor-associated antigen with a murine monoclonal antibody, LISA 101, selected by reversed indirect enzyme-linked immunosorbent assay.

In the presence of a characterized monoclonal antibody recognizing a soluble molecule, additional monoclonal antibodies reactive with unknown antigenic determinants on the molecule can be easily selected by reversed indirect enzyme-linked immunosorbent assay. A novel murine monoclonal antibody, LISA 101, was selected by reversed indirect enzyme-linked immunosorbent assay against soluble antigens, which exist in sera and in pleural effusions derived from lung adenocarcinoma patients and which bear determinants recognized by the previously characterized murine monoclonal antibody KL-6. Antigenic determinants recognized by the LISA 101 antibody appear to be sialylated carbohydrate in nature and different from those recognized by previously reported monoclonal antibodies against sialylated carbohydrates, such as NS 19-9, FH-6, and KL-6, suggested by competitive inhibition assay and immunostaining of tissues. A circulating antigen, LISA 1-6, was detected by a bimonoclonal bideterminant assay using immobilized LISA 101 antibody and enzyme-labeled KL-6 antibody. It was found that serum LISA 1-6 levels were elevated in 63% (25 of 40) of patients with lung adenocarcinoma and in 92% (11 of 12) of patients with pancreatic carcinoma, but only in 6.5% (2 of 31) of patients with benign lung diseases and in 7.1% (1 of 14) of patients with pancreatitis. The present observations indicate that the LISA 1-6 antigen may serve as a new tumor marker for adenocarcinomas of the lung and the pancreas. Additionally, the reversed indirect enzyme-linked immunosorbent assay may be a widely applicable method for selecting new monoclonal antibodies against as yet unknown antigenic determinants on soluble molecules.

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.

Prediction of short- and intermediate-term prognoses of patients with acute myocardial infarction using myocardial contrast echocardiography one day after recanalization.

OBJECTIVES: This study sought to determine whether microvascular integrity in the risk area (RA) for myocardial infarction (MI) one day after recanalization predicts the outcome in patients with first acute MI. BACKGROUND: Immediately after recanalization, microcirculation in the RA is modified by both hyperemic response and microvascular impairment. METHODS: Fifty consecutive patients who underwent serial myocardial contrast echocardiography before and one day after recanalization (day 2) were studied. All patients had a completely occluded lesion in the left anterior descending coronary artery alone, and underwent successful reperfusion therapy. The relative size of the initial RA (RA ratio) and peak gray scale ratio (PGSR) within the RA on day 2 were determined. Patients were followed for a median of 22 months to evaluate clinical outcome. RESULTS: On day 2, PGSR was a median of 0.46. Study patients were subdivided into two groups, group A of 24 patients with acceptable opacification (PGSR > 0.46 on day 2) and group B of 26 patients without it. Major cardiac events (cardiac death, nonfatal MI and repeat admission for congestive heart failure) were more frequently observed in group B (28% vs. 4%, Cox hazard ratio=8.5, p=0.05, 95% confidence interval [CI] 1.03 to 69.9). The median value of the RA ratio was 0.45. Patients (n=15) with RA ratio > 0.45 on day 1 and PGSR on day 2 < or = 0.46 exhibited a 10.7-fold relative risk for major cardiac events (p=0.005, 95% CI 2.06 to 55.8) and a 3.69-fold relative risk for composite cardiac events (major cardiac events and target lesion revascularizations) after the initial intervention (p=0.004, 95% CI 1.51 to 9.04). CONCLUSIONS: The assessment of both the size of the initial RA and microvascular integrity on day 2 enables precise determination of the efficacy of reperfusion therapy and prediction of the short- and intermediate-term prognoses of patients with recanalized MI.

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.

Carotid atherosclerosis and serum lipoprotein(a) concentrations in patients with NIDDM.

OBJECTIVE: To investigate the association of carotid atherosclerosis and serum lipoprotein(a) [Lp(a)] concentrations in subjects with NIDDM. RESEARCH DESIGN AND METHODS: We measured carotid intima-media thickness (IMT) and Lp(a) concentrations in 117 NIDDM subjects. Subjects were divided into tertiles according to IMT values and number of plaques. RESULTS: Serum Lp(a), but not lipid and apoprotein levels, increased significantly with increasing IMT (20.0 +/- 2.3, 24.7 +/- 3.3, and 39.8 +/- 4.3 mg/dl [mean +/- SE], respectively, P < 0.001). Serum Lp(a) increased with increasing number of plaques (18.4 +/- 2.5 mg/dl in 59 subjects with no plaques, 25.8 +/- 2.5 mg/dl in 24 subjects with 1 plaque, and 38.7 +/- 5.1 mg/dl in 34 subjects with more than 1 plaque; P < 0.05). Furthermore, the mean IMT and Lp(a) levels in the subjects with cerebrovascular disease (CD) were significantly higher than in those without CD (1.25 +/- 0.04 mm and 41.2 +/- 4.7 mg/dl vs. 1.08 +/- 0.03 mm and 22.2 +/- 1.9 mg/dl; P < 0.005). The mean IMT and Lp(a) levels were higher in subjects with ischemic heart disease (IHD) than in those without IHD, although statistical significance was not observed (1.21 +/- 0.06 mm and 31.7 +/- 4.7 mg/dl vs. 1.10 +/- 0.03 mm and 27.0 +/- 2.4 mg/dl, respectively). CONCLUSIONS: Elevated serum Lp(a) concentrations are associated with carotid atherosclerosis in NIDDM subjects.

Dissociation of microangiopathy and macroangiopathy in patients with type 2 diabetes.

OBJECTIVE: Although persistent hyperglycemia contributes greatly to the progression of diabetic micro- and macroangiopathy, microangiopathy progresses more rapidly than macroangiopathy in some type 2 diabetic patients, with the opposite being true in others. This study was conducted to identify factors responsible for such dissociation. RESEARCH DESIGN AND METHODS: Patients with proliferative diabetic retinopathy and a carotid intima-media thickness (IMT) level < or =1.0 mm were classified as the microangiopathy group (MIG); those with an IMT level >1.1 mm and without retinopathy or with background retinopathy were assigned to the macroangiopathy group (MAG). Only middle-aged patients, 50-69 years old, were included in this study. There were 54 patients in the MIG and 68 patients in the MAG. RESULTS: Patients in the MIG were significantly younger at the onset of diabetes, and those in the MAG had a significantly higher mean ratio of apoprotein (apo) B to apoAI. The percentage of patients with a family history of diabetes was significantly higher in the MIG. Maternal inheritance was common among these patients. Those with obesity, a family history of diabetes, and younger onset of hypertension were more common in the MAG. In the multiple logistic regression analyses, maternal inheritance and early onset of diabetes were independent risk factors for the acceleration of microangiopathy. A personal history of obesity and a family history of hypertension were independently related to the development of macroangiopathy. CONCLUSIONS: Our results suggest that patients with early onset and maternal inheritance of diabetes may have a high risk for the progression of diabetic microangiopathy, while patients with hyperlipidemia, a history of obesity, and a family history of hypertension seem prone to the development of atherosclerosis.

Diabetes mellitus and its vascular complications in Japanese migrants on the Island of Hawaii.

Japanese migrants and their offspring on the island of Hawaii and Japanese living in Hiroshima were examined for diabetes mellitus and its vascular complications. the same methods and investigators were used in both locations. Death certificates of Japanese and Caucasians dying on the island during the past 26 yr were analyzed. Diabetes, defined as a venous serum glucose concentration of at least 200 mg/dl 2 h after a 50-g oral glucose load, was significantly more common in the Hawaiian Japanese than in the Hiroshima Japanese subjects. This suggests that diabetes is more prevalent in Japanese in Hawaii than in Japan, although lack of knowledge about the total population of Japanese migrants in Hawaii makes this generalization uncertain. The proportion of deaths attributed to diabetes was much higher in Japanese migrants and their offspring in Hawaii than in Japan. During the 1950s, the proportional death rate from diabetes was about half as large in Japanese Hawaiians as in Caucasian Hawaiians, but it increased to become 1.6 times the Caucasian rate during the 1970s. A nutritional study revealed that the total caloric intake was similar in Japanese in Hawaii and Hiroshima, although the estimated level of physical activity was less in the Hawaiian subjects. Consumption of animal fat and simple carbohydrates (sucrose and fructose) were at least twice as high in Hawaiian as in Hiroshima Japanese. Conversely, Hiroshima Japanese consumed about twice the amount of complex carbohydrate as the Hawaiian Japanese. These observations support the hypothesis that a high fat, high simple carbohydrate, low complex carbohydrate diet and/or reduced levels of physical activity increase risk of diabetes. The proportion of deaths attributed to ischemic heart disease was higher in both diabetic and nondiabetic Japanese Hawaiians than in diabetic subjects in Japan. The rates were similar for Japanese and Caucasians in Hawaii. There was no evidence of an environmental influence on the development of microangiopathy (retinopathy) in diabetes, as the prevalence of diabetic retinopathy (stratified for diabetes duration) was similar in Japanese subjects in Hawaii and in Japan, and it was similar to previous reports from England. On the other hand, diabetes alone did not appear to account for the greater prevalence of macroangiopathy in Hawaiian Japanese than in Hiroshima. Thus environmental factors, possibly including diet, appear to be involved in the development of macrovascular complications of diabetes.

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.