Characterization of PacMetUT1, a recently isolated human prostate cancer cell line.

BACKGROUND: Existing prostate cancer cell lines have limitations. METHODS: Cells were characterized using Western blotting, immunohistochemistry, invasion into Matrigel, and by studying xenograft tumors. RESULTS: We describe a cell line (PacMetUT1) isolated from a lymph node of a 57-year-old male with prostate cancer. Compared to existing prostate cancer cell lines, the growth rate of PacMetUT1 xenograft tumors is slower with tumors occurring at injection sites and with metastases to lung and liver. Androgen receptor (AR) was detected in vivo by Western blotting and the cells responded to methyltrienolone (R1881). PacMetUT1 cells are more invasive in Matrigel than DU-145, PC-3, and LNCaP cells, and showed greater anchorage-independent growth in soft agar. The cells do not express prostate specific antigen (PSA) in vitro or in xenografts. However, the green fluorescent protein (GFP) gene was introduced and stably expressed in PacMetUT1 cells, allowing tumor imaging in vivo. Xenograft tumors show epithelial features and are positive for keratin, epithelial membrane antigen, EGF receptor, and E cadherin. In contrast, fibroblast markers vimentin, desmin, and Factor VIII, were negative. Karyotyping showed losses of 6p, 7q, 8p, 18q, and 22q, and gains of 8q and 9q; additional genetic material was observed at 2q and 12p. CONCLUSION: The PacMetUT1 cell line allows metastases to be assessed using a single animal model. Because of its slower growth, PacMetUT1 more closely mimics the human disease. Studies of tumor progression or metastasis can be conducted over a longer period of time.

Familial supernumerary marker chromosome evolution through three generations.

A mosaic chromosome complement, 46,XY/47,XY,+r(15), was detected at prenatal diagnosis. Family studies showed the mother and one of her two children to have a bisatellited supernumerary marker chromosome (SMC) in all lymphocytes examined. The maternal grandfather also showed a bisatellited SMC, but in only 2 per cent of his lymphocytes. NOR, DA/DAPI, and chromosome 15 centromere and short arm-specific probes confirmed the identify of the bisatellited SMC and of ring SMC as derived from chromosome 15. An apparently normal male was born at full term. At age 1 year, the baby continues to have normal growth and development. The bisatellited 15 likely originated by somatic mutation in the grandfather (2 per cent cells), was transmitted unchanged to the daughter and grandson (germline transmission, no mosaicism), and then evolved by excising the satellites and forming a ring SMC in the index case. Progressive changes in the frequency and subsequent changes in the structure of this SMC illustrate the unusual characteristics of chromosome 15.

Discrepancies in cytogenetic findings in chorionic villi.

We present a database and a review of published literature on discrepancies in chorionic villus (CV) diagnostic findings. The review includes 457 cases of discrepancies between CV findings (direct, culture, or both) and the fetus. One hundred and one cases reported normal by CV direct harvest included 30 with abnormal or mosaic abnormal fetal karyotype (30% false negatives). The corresponding number of false negatives for CV culture was only 4 cases out of 133 (3%). Assuming no bias in reporting cases based on site of discrepancy (assumption may not hold), these data imply that the probability of false-negative findings is 10-fold higher by CV direct method compared to CV culture method. We also reviewed recent studies reporting on large series of CV diagnosis. This review revealed that the reported overall frequencies of discrepancies as a percentage of abnormal and mosaic abnormal CV results ranged from 11 to 63% (a mean of 37%). These data, together with recent reports of survival of embryos with reported abnormal karyotypes because of confined placental mosaicism (CPM), raise several questions pertaining to the predictive value of CV sampling and the origin of the discrepancies in the fetal-placental unit. Caution is recommended in counseling patients undergoing CV sampling to provide appropriate follow-up studies in cases of possible discrepancies.

Elevated mutagen susceptibility in cultured lymphocytes of oral cancer patients.

Mitomycin C (MMC)-induced lymphocytic sister chromatid exchange (SCE) frequency was studied in 40 oral cancer (OC) patients, 40 normal tobacco chewers (NC) and in 40 normal healthy individuals not consuming tobacco/areca nut in any form. Significantly higher MMC-induced SCE/cell values were observed among OC patients as compared to healthy non-chewer controls as well as NC. Although the mean SCE frequency for NC was comparable to that of healthy controls, three individuals showed an SCE rate higher than the highest observed among controls. The comparable frequency of the tobacco habit in these three individuals with that of the rest of the thirty-seven individuals indicated the possible involvement of factors other than tobacco consumption for the higher susceptibility to mutagens.

Urine of tobacco/areca nut chewers causes genomic damage in Chinese hamster ovary cells.

The chromosome-damaging effects of urine concentrates (UCs) from tobacco plus areca nut (T/AN) chewers (a highly popular habit and a major risk factor for oral cancer in India) were evaluated on Chinese hamster ovary (CHO) cells employing two cytogenetic end-points, namely chromosome aberration (CA) and sister chromatid exchange (SCE) frequencies. Urine creatinine levels were comparable between controls and T/AN chewers. CA and SCE frequencies in CHO cells were found to be elevated significantly (P < 0.001) following treatment with UCs prepared from T/AN chewers (UC-T/AN chewers) as well as with UCs of non-chewer controls (UC-control subjects). Moreover, elevation of these two parameters by UC-T/AN chewers was significantly higher in comparison to that of UC-controls. The results of the present study indicated that besides the oral cavity, which is a target organ for T/AN chewers, mutagens/carcinogens in tobacco and areca nut might be playing a causative role in cancer of the urinary bladder as well.

Ethanol potentiates the clastogenicity of pan masala--an in vitro experience.

The significance of the interaction between alcohol and tobacco in causing head and neck cancers is well documented. Our previous reports on in vitro studies using aqueous and organic extracts as well as cytogenetic studies among pan masala consumers have conclusively shown the genotoxic potential of pan masala--a dry mixture of the areca nut, lime, catechu, unspecified flavouring agents, etc., often containing tobacco in it and is widely consumed in India. Now in the present report, the clastogenic effect of ethanol and pan masala in different combinations was evaluated on Chinese hamster ovary cells utilizing chromosome aberration (CA) frequency as an endpoint. An ethanol concentration of up to 2.0% had no effect on CA/cell value. The low-dose continuous treatment and high-dose short-term pre-, post- and simultaneous treatment of ethanol and aqueous extract of pan masala with and without tobacco yielded dose-dependent elevations in CA frequency, compared to any of these two substances alone. Thus, these results provide evidence that alcohol consumption may potentially increase the risk of oral cancer among pan masala chewers.

Clinical application of serum levels of sialic acid, fucose and seromucoid fraction as tumour markers in human leukemias.

To establish a blood-based biochemical profile for diagnosis and management of human leukemias, serum total sialic acid/total protein, lipid bound sialic acid, fucose/total protein and seromucoid fraction (quantitated as its hexoses and protein contents) levels were measured by highly specific spectrophotometric methods. Compared to the controls (n = 150), all the biomarkers were significantly elevated in anemia patients (pathological controls, n = 77) and untreated leukemia patients (n = 145). Furthermore, significantly raised levels of the markers were observed in untreated leukemia patients compared to anemia patients. Fucose/total protein was the most specific (71.0%) marker, while hexose levels were the most sensitive (93.0%) marker for leukemia. The levels of the biomarkers in patients with persistent leukemic activity/accelerated leukemic phase were significantly higher than those in patients in remission and were comparable with pretreatment levels. The study suggested that the markers evaluated are useful in diagnosis and treatment monitoring of leukemia patients.

Pan masala--a genotoxic menace.

Cytogenetic markers such as chromosome aberration (CA), sister-chromatid exchange (SCE) and micronucleated cells (MNC) were used to assess the genotoxic potential of dimethyl sulphoxide (DMSO) extract of pan masala with and without tobacco (PM-T and PM). Using in vitro short-term assays, the extracts were tested in the presence or absence of metabolic activation. In cultures without metabolic activation the extracts were found to increase the frequency of all the three parameters tested significantly, however those with activation elicited a weak response, implying that pan masalas contain solvent (DMSO)-soluble direct-acting mutagen.

Evaluation of chemopreventive effects of betel leaf on the genotoxicity of pan masala.

The antigenotoxic effect of the aqueous extract of betel leaf (BL-ext.) against the pan masala was tested with the help of cytogenetic endpoints like chromosome aberration (CA) and sister chromatid exchange (SCE) utilizing Chinese hamster ovary (CHO) cells. Compared to the cultures treated with aqueous extract of pan masala alone, a reduction in CA and SCE frequencies in CHO cells was observed following a combined treatment with pan masala (with or without tobacco) extract and BL-ext. The protective effect of BL-ext. against the genomic damage caused by pan masala was statistically significant only after treating the cells for a longer period.

Serum lactate dehydrogenase and its isoenzymes in leukemia patients: possible role in diagnosis and treatment monitoring.

Serum levels of total LDH (T.LDH) and LDH isoenzymes in 145 untreated leukemia patients were examined and compared with those of 150 age and sex matched healthy individuals (controls) and 77 anemia patients (pathological controls). As compared to the controls, T.LDH, LDH-1, LDH-2, LDH-3, LDH-4 and LDH-5 values were significantly elevated (p < 0.001) in untreated leukemia patients. T.LDH, LDH-1, LDH-2 and LDH-3 levels were significantly raised in anemia patients as compared to the controls. A significant increase in the levels of T.LDH, LDH-2, LDH-3 and LDH-4 was observed in untreated leukemia patients as compared to the anemia patients. LDH-4 was most specific (81.0%), while LDH-2 was the most sensitive (84.0%) marker for leukemia. Mean values of all the markers were higher in leukemia patients with persistent leukemic activity/accelerated leukemic phase as compared to the patients in remission. Serum levels of T.LDH as well as all the five isoenzymes were significantly lower in leukemia patients in remission as compared to untreated leukemia patients. The study indicated that alterations in T.LDH and its isoenzymes are useful for diagnosis and treatment monitoring of leukemia.

Monitoring of smokeless tobacco consumers using cytogenetic endpoints.

Smokeless tobacco consumption is causally associated with oral cavity cancers; however, extensive cytogenetic studies have not been done. In the present study, individuals consuming dry snuff or tobacco with lime have been studied for frequency of micronucleated cells (MNC) in exfoliated buccal mucosa and chromosome aberrations (CA) and sister chromatid exchanges (SCE) in lymphocytes. The significant elevation in the values of all the three cytogenetic markers among tobacco users compared to the controls reveal the extent of genomic damage on target and nontarget tissues. The findings emphasize the possible use of cytogenetic endpoints for monitoring smokeless tobacco consumers.

Clinical significance of serum total and heat-stable alkaline phosphatase in leukemia patients.

AIMS AND BACKGROUND: Variations in serum alkaline phosphatase (ALP) and placental-like (heat stable) alkaline phosphatase (PLAP) are found to be clinically useful in cancer patients. The present study evaluated serum ALP and PLAP levels to establish a blood-based biochemical index for leukemia patients. STUDY DESIGN: ALP and PLAP levels were determined in 145 untreated leukemia patients, 77 anemia patients, 150 healthy individuals (controls), 47 leukemic patients in remission and 23 patients with persistent leukemic activity/accelerated leukemic phase (P. Leu./A. Leu.). The enzymes were estimated with highly specific spectrophotometric methods. RESULTS: Serum ALP and PLAP levels were significantly elevated in anemia patients and leukemia patients compared to controls. Comparison between anemia patients and leukemia patients showed insignificant differences for ALP, whereas PLAP levels were significantly raised (p < 0.001) in leukemia patients. ALP showed insignificant difference between untreated leukemia patients, patients with P. Leu./A. Leu. and leukemia patients in remission. PLAP levels were comparable in patients with P. Leu./A. Leu. and were significantly lower (p < 0.02) in leukemia patients in remission than in untreated leukemia patients. CONCLUSIONS: The results indicate that serum PLAP levels are a useful biochemical marker for diagnosis and treatment monitoring of leukemia patients. However, serum ALP levels have limited utility for leukemia patients.

Genotoxic effects of tobacco extract on Chinese hamster ovary cells.

Genotoxic effects of an aqueous extract of Nicotiana tabacum, a variety commonly used in India for chewing purposes, were analysed on CHO cells utilizing two different cytogenetic end-points, namely, chromosome aberration frequency and sister chromatid exchange frequency. Statistically significant elevations in the values of both the markers clearly indicated chromosome damaging effects of the extract. Elevations in chromosome aberration and sister chromatid exchange frequencies are suggestive of intrastrand and interstrand DNA cross-links following exposure to tobacco. The effects observed following treatment with low dose for longer duration are of relevance to the condition of the oral mucosa of the chronic smokeless tobacco users.

Incidence of micronuclei in oral mucosa of users of tobacco products singly or in various combinations.

Frequencies of micronucleated cells (MNCs) were analyzed in the exfoliated buccal mucosa of normal healthy individuals from different parts of India who were regularly using either areca nut alone, mava, tamol, tobacco with lime, dry snuff or masheri. The analyses were also carried out among oral submucous fibrosis patients who had the habit of chewing either mava or areca nut. Compared with 'no habit' healthy individuals, all the groups, irrespective of their type of habit, had significantly higher frequencies of MNCs.

Role of areca nut consumption in the cause of oral cancers. A cytogenetic assessment.

BACKGROUND: Cytogenetic studies, framed to assess the possible genomic damage caused by areca nut consumption (without tobacco and not as a component of betel quid), were performed among areca nut chewers, which included normal people who chew areca nuts, patients with oral submucous fibrosis, and patients with oral cancer, and healthy nonchewing controls. RESULTS: The analysis showed statistically significant increases in the frequencies of sister chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and the percentage of micronucleated cells in exfoliated cells of buccal mucosa among all three groups of chewers when compared with those of the controls. CONCLUSIONS: The current data, the first of this type among only areca nut chewers, highlight that this popular masticatory is erroneously considered "safe" and that it increases the genomic damage even when chewed without tobacco. The data also signify that, henceforth, in cytogenetic biomonitoring, areca nut consumption also should be considered as one of the confounding factors.

Chromosome damaging effects of pan masala.

Effects of aqueous extracts of a popular brand of pan masala with and without tobacco (PM-T and PM) were studied for short duration treatment employing an in vitro system. Metabolic activation with S9 mix was also included. Frequency of all the three cytogenetic endpoints viz., chromosome aberration (CA); sister chromatid exchange (SCE) and % micronucleated cells (% MNC) were found to be elevated significantly in a dose-dependent manner in cultures without metabolic activation. However, addition of S9 activation system resulted in suppression of chromosomal damage. Our findings indicate that pan masalas contain water soluble direct acting mutagens.

In vitro genotoxic effects of areca nut extract and arecoline.

The genotoxic potential of the aqueous extract of areca nut as well as arecoline, the major alkaloid of the areca nut, was tested with the help of cytogenetic markers such as sister-chromatid exchanges and chromosome aberrations, utilizing Chinese hamster ovary (CHO) cells. The continuous-treatment and pulse-treatment schedules yielded dose-dependent elevations in the frequencies of sister-chromatid exchange and chromosomal aberration in CHO cells, indicating a genotoxic effect of both the extract and arecoline. The results also imply that, besides arecoline, there may be some other water-extractable substances in the areca nut that make the extract more genotoxic. The chromosome damage was found to be more severe on treating the cells with low concentrations and for longer duration, which mimic the effects of chronic areca nut consumption.

Cytogenetic surveillance of tobacco-areca nut (mava) chewers, including patients with oral cancers and premalignant conditions.

Three cytogenetic endpoints were studied in non-chewing healthy controls and 3 groups of tobacco-areca nut chewers, viz. normal chewers, chewers with oral submucous fibrosis and chewers with oral cancer. Frequencies of sister-chromatid exchanges and chromosome aberrations in peripheral blood lymphocytes and of micronucleated cells in exfoliated buccal mucosa were evaluated. All the parameters showed statistically significant elevations in all 3 groups of chewers compared to the controls. The data indicate possible application of the parameters as sensitive endpoints for monitoring tobacco-areca nut chewers, the group of individuals at higher risk of developing oral cancer, the commonest cancer among Indian males.

Tumor markers in leukemia: evaluation of serum levels of different forms of sialic acid, Regan isoenzyme and lactate dehydrogenase.

A sensitive and specific serum marker can greatly help in the early diagnosis of malignancy as well as in monitoring the treatment of cancer patients. The present work was initiated for determining serum levels of Total Sialic Acid (TSA), Lipid Bound Sialic Acid (LSA), Free Sialic Acid (FSA). Regan Isoenzyme (RI) and Lactate Dehydrogenase (LDH), so as to evaluate their value as potential tumor markers. Fifty patients with anemia and 78 patients with leukemia were studied. The leukemia group consisted of 32 cases of Acute Myeloid Leukemia (AML), 29 cases of Chronic Myeloid Leukemia (CML) and 17 cases of Acute Lymphatic Leukemia (ALL). The levels were compared with the values obtained from 88 healthy individuals. Compared to the healthy controls, all the biomarkers were significantly elevated in patients with anemia as well as in those with leukemia. However, in leukemia patients significantly higher levels of TSA, LSA, FSA and LDH were observed compared to anemia patients. TSA levels were significantly higher in AML patients compared to CML and ALL patients. LSA levels were also significantly higher in AML patients compared to ALL patients. LSA was the most sensitive (84.6%) while FSA and RI levels were the most specific (78.0%) markers for leukemia. The combined use of the markers showed increased sensitivity and specificity (100.0% and 98.0%, respectively). The study suggested that the biomarkers investigated might be used for differentiating anemic from leukemic conditions, however, more in-depth studies are indicated to assess their utility in classifying various leukemias.