Comparison of high dose rate, low dose rate, and high dose rate fractionated radiation for optimizing differences in radiosensitivities in vitro

In an attempt to clarify the biological nature of a human endogenous retrovirus (HERV), HERV-R, which is a single-copy type of HERVs and is conserved as a full-length viral sequence, the expression of HERV-R mRNA in normal autopsied systemic organs was examined by Northern blot analysis. The expression showed different levels among individuals, with the adrenal glands expressing the highest level of HERV-R among all organs tested, except for the placenta. In various adrenal tumors, HERV-R was expressed at high levels in all cortical adenomas but less so in pheochromocytomas. In situ hybridization revealed the expression of HERV-R to be localized in all layers of the adrenal cortex, but not in the medulla. This high-level expression of HERV-R in the adrenal cortex may possibly relate to differentiation and/or steroid production by adrenocortical cells.     

Chemosensitization of human prostate carcinoma cell lines to anti-fas-mediated cytotoxicity and apoptosis

Androgen ablation has been an effective treatment in patients with advanced prostate cancer. However, most treated patients develop hormonally resistant disease and do not respond to conventional chemotherapy. Immunotherapy against prostate cancer is an alternative approach in overcoming hormonal/drug-resistant prostate cancer. Cytotoxic immune lymphocytes kill target cells via the perforin/granzyme and the Fas-ligand (Fas-L) pathways. We hypothesize that tumor cells respond poorly to immunotherapy by developing resistance to killing by the Fas-L mechanism. This study investigated whether prostate tumor cells are sensitive to Fas-mediated killing. The human prostate carcinoma cell lines DU145, PC-3, and LnCAP were examined for their sensitivity to killing and apoptosis by the Fas-L agonist anti-Fas antibody and CTLs. All three lines moderately expressed the Fas antigen on the cell surface; however, all three lines were relatively resistant to cytotoxicity mediated by anti-Fas (CH-11) antibody. Pretreatment of DU145 and PC-3 with subtoxic concentrations of drugs followed by anti-Fas antibody resulted in synergistic cytotoxicity and apoptosis, whereas only an additive effect was obtained with LnCAP. Chemosensitization with drugs and anti-Fas was completely blocked by the addition of neutralizing anti-Fas antibody. The murine CTL hybridoma, PMMI, which kills only via the Fas-L pathway, was able to kill chemosensitized PC-3 and DU145 but not LnCAP cells. Furthermore, this cytotoxicity was blocked by anti-Fas neutralizing antibody. Chemosensitization of PC-3 and DU145 prostate tumor cells was not due to up-regulation of Fas-receptor antigen expression. Treatment of tumor cells with cisplatin did not down-regulate the antiapoptotic genes bcl-2, FAP-1, and c-myc. Further, there was no induction by cisplatin of Fas-L on the tumor cells, thus ruling out Fas/Fas-L-mediated autologous killing. These findings demonstrate that pretreatment of drug-resistant/CTL-resistant prostate DU145 and PC-3 tumor cells with subtoxic concentrations of certain chemotherapeutic drugs sensitizes the tumor cells to Fas-mediated cytotoxicity. These findings suggest that chemosensitization of tumor cells should optimize the response to immunotherapeutic interventions in the treatment of hormone-resistant/drug-resistant prostate cancer.     

Establishment and characterization of human gastric carcinoma cell lines

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.     

Long-term health effects of low dose exposure to nerve agent

Possible long-term toxic effects of nerve agents have been investigated using sensitive toxicological screens and extensive toxicity studies in various animal models. Data on humans have been obtained from controlled studies and accidental exposures. Studies in the area of 'low dose' exposure to nerve agents are currently being performed.     

Assessment of exposure rate and collective effective dose equivalent in the city of Baghdad due to natural gamma radiation

Exposure rate measurements in the city of Baghdad were initiated in June 1981. The average exposure rate, the average annual effective dose equivalent and the collective effective dose equivalent are assessed. The data are presented according to the eleven municipal divisions of the city. The average exposure rate was 6.9 microR h-1 and an average annual effective dose equivalent of 4.5 x 10(-4) Sv year-1. The collective effective dose equivalent is 1.81 x 10(3) man Sv year-1.     

Expression of human prostatic acid phosphatase correlates with androgen-stimulated cell proliferation in prostate cancer cell lines

Androgen plays a critical role in regulating the growth and differentiation of normal prostate epithelia, as well as the initial growth of prostate cancer cells. Nevertheless, prostate carcinomas eventually become androgen-unresponsive, and the cancer is refractory to hormonal therapy. To gain insight into the mechanism involved in this hormone-refractory phenomenon, we have examined the potential role of the androgen receptor (AR) in that process. We have investigated the expression of AR and two prostate-specific androgen-responsive antigens, prostatic acid phosphatase (PAcP) and prostate-specific antigen (PSA), for the functional activity of AR in LNCaP and PC-3 human prostate carcinoma cells. Our results are as follows. (i) Clone 33 LNCaP cells express AR, PAcP, and PSA, and cell growth is stimulated by 5alpha-dihydrotestosterone (DHT). Stimulation of cell growth correlates with decreased cellular PAcP activity. (ii) In clone 81 LNCaP cells, the expression of PAcP decreases with a concurrent decrease in the degree of androgen stimulation of cell growth, whereas the expression of PSA mRNA level is up-regulated by DHT, as in clone 33 cells. Conversely, in PAcP cDNA-transfected clone 81 cells, an additional expression of cellular PAcP correlates with an increased stimulation by androgen, higher than the corresponding control cells. (iii) PC-3 cells express a low level of functional AR with no detectable PAcP or PSA, and the growth of PC-3 cells is not affected by DHT treatment. Nevertheless, in two PAcP cDNA-transfected PC-3 sublines, the expression of exogenous cellular PAcP correlates with androgen stimulation. This androgen stimulation of cell growth concurs with an increased tyrosine phosphorylation of a phosphoprotein of 185 kDa. In summary, the data indicate that the expression of AR alone is not sufficient for androgen stimulation of cell growth. Furthermore, in AR-expressing prostate cancer cells, the expression of cellular PAcP correlates with androgen stimulation of cell proliferation.     

Inhibition of the mitogen activated protein (MAP) kinase cascade potentiates cell killing by low dose ionizing radiation in A431 human squamous carcinoma cells

The molecular mechanism(s) by which tumor cells survive after exposure to ionizing radiation are not fully understood. Exposure of A431 cells to low doses of radiation (1 Gy) caused prolonged activations of the mitogen activated protein (MAP) kinase and stress activated protein (SAP) kinase pathways, and induced p21(Cip-1/WAF1) via a MAP kinase dependent mechanism. In contrast, higher doses of radiation (6 Gy) caused a much weaker activation of the MAP kinase cascade, but a similar degree of SAP kinase cascade activation. In the presence of MAP kinase blockade by the specific MEK1 inhibitor (PD98059) the basal activity of the SAP kinase pathway was enhanced twofold, and the ability of a 1 Gy radiation exposure to activate the SAP kinase pathway was increased approximately sixfold 60 min after irradiation. In the presence of MAP kinase blockade by PD98059 the ability of a single 1 Gy exposure to cause double stranded DNA breaks (TUNEL assay) was enhanced at least threefold over the following 24-48 h. The increase in DNA damage within 48 h was also mirrored by a similar decrease in A431 cell growth as judged by MTT assays over the next 4-8 days following radiation exposure. This report demonstrates that the MAP kinase cascade is a key cytoprotective pathway in A431 human squamous carcinoma cells which is activated in response to clinically used doses of ionizing radiation. Inhibition of this pathway potentiates the ability of low dose radiation exposure to induce cell death in vitro.     

Angiogenic factors expressed by human prostatic cell lines: effect on endothelial cell growth in vitro

Using anterograde transport of WGA-HRP and the experimental degeneration method for identification of corticocuneate (CCT) and primary afferent (PAT) terminals in conjunction with gamma-amino butyric acid (GABA) and glutamate immunocytochemistry, this study has demonstrated that the GABA-immunoreactive (GABA-IR) neurons in the rat cuneate nucleus were post-synaptic to PATs (some of them being glutamate-IR), GABA-IR and GABA-negative terminals. The HRP-labelled CCTs did not make any synaptic contacts with GABA-IR neurons but with some GABA-negative dendrites. PATs labelled by HRP or showing degenerating features made direct synaptic contacts with the dendrites of GABA-IR neurons. Beside the above GABA-IR boutons also showed axosomatic and axodendritic synapses with the GABA-IR neurons. In 'triple labeling' method for GABA, PAT and glutamate, it was found that the PATs which were usually glutamate-positive were presynaptic to the dendrites of GABA-IR neurons. Furthermore, some glutamate-IR terminals which were of non-PAT's origin also synapsed with the dendrites and somata of GABA-IR neurons. It is concluded from this study that the major inputs of GABA-IR neurons were from glutamate immunopositive PATs and glutamate terminals of non-PATs origin; other GABA-IR terminals either intrinsic or extrinsic also contributed to the afferent sources of GABA-IR neurons. The CCTs contributed very little, if any, to this input. It is suggested that the PATs and glutamate-IR terminals on GABA-IR neurons may be involved in lateral inhibition for increase of spatial precision. The synaptic contacts between GABA-IR boutons and dendrites or somata of GABA-IR neurons may provide a possible means for disinhibition.     

Endothelin expression and responsiveness in human ovarian carcinoma cell lines

To elucidate the potential role of endothelins (ETs) as growth regulators in ovarian carcinoma cells in culture, expression of endothelins and their receptors were measured in two ovarian cancer cell lines (PEO4 and PEO14), together with the effect of the exogenous addition of endothelins on the growth of these cell lines in vitro. RT-PCR analysis of mRNA prepared from PEO4 and PEO14 indicated the presence of ET-1 and ET-3 mRNA. Immunoreactive ET-1-like peptide was found in media from cultures of both PEO4 (1.7 +/- 0.4 fmol/10(6) cells/72 h) and PEO14 (20.2 +/- 6.8 fmol/10(6) cells/72 h) cell lines. Radioligand binding studies using 125I-ET-1 and membrane fractions were consistent with PEO4 cells having two receptor sites of either high affinity (Kd = 0.065 nM, Bmax = 0.047 pmol/mg protein) or lower affinity sites (Kd = 0.49 nM, Bmax = 0.23 pmol/mg protein). Studies using membrane fractions of PEO14 cells indicated that this cell line has only a single lower affinity binding site (Kd = 0.56 nM, Bmax = 0.31 pmol/mg protein). However, RT-PCR analysis indicated the presence of mRNA from both ETA and ETB receptors in PEO4 and PEO14 cell lines. Exogenous addition of ETs to PEO4 and PEO14 cells at concentrations of 10(-10)-10(-7)M resulted in specific dose-dependent increases in cell number for ET-1 (with maximum effects at 10(-10) and 10(-9)M for PEO4 and PEO14, respectively) and ET-2 (maximum effects at 10(-8) and 10(-9)M for PEO4 and PEO14, respectively) but not for ET-3. Experiments on the growth of PEO14 cells using BQ123 (ETA-R) antagonist and "antisense" oligonucleotide against the ETA-R, in the absence of exogenous ETs, suggested that immunoreactive ET-1-like material secreted by PEO14 cells can affect their growth in an autocrine manner. These results would be consistent with ET-1 acting as a possible autocrine growth regulator in human ovarian carcinoma cells.     

Factors affecting mandibular complications in low dose rate brachytherapy for oral tongue carcinoma with special reference to spacer

PURPOSE: To evaluate the efficacy of a spacer in the prevention of mandibular complications in low dose rate (LDR) brachytherapy (BRT) for oral tongue carcinoma. METHODS AND MATERIALS: A retrospective analysis was conducted using 103 patients with T1 or T2 tongue carcinoma treated by a single plane implantation of iridium (192Ir) pins between 1979-1994. Of these patients, 60 were treated by BRT alone, and the rest were combined with external irradiation (Ext) and/or chemotherapy (CHT). Forty-eight and 55 patients were given BRT with and without a spacer, respectively. Spacers were individually made of acrylic resin according to a prosthetic technique so as to obtain the thickness of 7-10 mm at the lingual part of the implanted side. Variables, including a spacer, which may be associated with the development of osteoradionecrosis (ORN) of the mandible, were analyzed by the Cox proportional hazards regression analysis. RESULTS: Our spacer reduced about 50% of the absorbed dose at the lingual side surface of the lower gingiva (LSG) to that in the absence of a spacer. Absolute incidence of ORN was 2.1% (1 of 48) and 40.0% (22 of 55), with and without a spacer, respectively, and the difference was statistically significant by univariate analysis (p = 0.0004). It was revealed by the Cox analysis that the spacer (p = 0.0247), combined CHT (p = 0.0295), and combined Ext (p = 0.0279) were significant independent factors associated with the development of ORN. The spacer was shown to be a significant factor by univariate analysis (p = 0.0037), but not by multivariate analysis when analysis was restricted to the patients who did not receive CHT. The absorbed dose, dose rate, and biological effective dose (BED) reflecting early or late response were estimated at the LSG, and prognosticators associated with the incidence of ORN were also determined by the Cox analysis. Particularly, BED for late response by BRT, the total absorbed dose, and any BED by Ext plus BRT were highly significant factors in the whole population. Essentially similar results were obtained in the patients without receiving CHT. CONCLUSIONS: It was clarified that our spacer effectively prevents mandibular complications in LDR BRT by 192Ir for oral tongue carcinoma. Furthermore, introduction of a spacer provided novel information concerning the development of ORN, where BED particularly for late response given by BRT, the total absorbed dose, and any BED by Ext plus BRT could be good prognostic factors only when estimated at the LSG.     

Establishment of xenografts and cell lines from well-differentiated human thyroid carcinoma

OBJECTIVE: To investigate the acute effect of cigarette smoking on glucose tolerance, insulin sensitivity, serum lipids, blood pressure, and heart rate. RESEARCH DESIGN AND METHODS: This nonrandomized experimental control trial in a tertiary care center included 20 healthy chronic smokers and 20 age-, sex-, and BMI-matched healthy volunteers. Two oral glucose tolerance tests (OGTTs) were performed on each subject. Three cigarettes were smoked during the first 30 min in one of the tests. Serum glucose, insulin, and C-peptide levels were measured every 30 min; the area under the curve (AUC) and the insulin sensitivity index (ISI) were calculated; serum total cholesterol, LDL cholesterol, HDL cholesterol, and triglyceride levels were measured at 0 and 180 min; and blood pressure and heart rate were recorded every 5 min throughout 180 min. RESULTS: Smoking acutely impaired glucose tolerance: the AUC for glucose in smokers was 25.5 +/- 1.03 mmol/l (mean +/- SE) (95% CI 22.9-28) during the smoking OGTT and 21.8 +/- 0.85 mmol/l (CI 19.2-24.3) in the control OGTT (P < 0.01); in nonsmokers, it was 19.7 +/- 0.3 mmol/l (CI 18.8-20.5) in the smoking OGTT and 18.7 +/- 0.35 mmol/l (CI 17.8-19.5) in the control OGTT (P < 0.05). Smoking acutely increased serum insulin and C-peptide levels and decreased ISI only in smokers: ISI in smokers was 55 +/- 2.8 (CI 47.4-62.6) in the control OGTT and 43 +/- 2.7 (CI 35.4-50.6) in the smoking OGTT (P < 0.05). Smoking acutely caused a rise of serum total cholesterol levels in both groups and increased LDL cholesterol and triglyceride serum levels significantly only in smokers (P < 0.05). A significant rise of blood pressure and heart rate while smoking was present in all the subjects. CONCLUSIONS: Smoking acutely impaired glucose tolerance and insulin sensitivity, enhanced serum cholesterol and triglyceride levels, and raised blood pressure and heart rate. These findings support the pathogenetic role of cigarette smoking on cardiovascular risk factors.     

Etoposide sensitivity of human prostatic cancer cell lines PC-3, DU 145 and LNCaP

Metastatic prostatic cancer is typically refractory to androgen ablation therapy due to the presence of androgen-independent clones in the neoplasia. A therapeutical approach which could effectively control androgen-dependent and independent cells is, thus, needed. Maybe the failure of certain cancer cells to engage in apoptosis could explain the inherent drug resistance of many tumors. Anyway, these cells can retain the ability to undergo apoptosis in response to an adequate stimulus. We tested whether etoposide, a topoisomerase II inhibitor, could induce apoptosis in androgen-dependent (LNCaP) as well as independent (PC-3 and DU 145) human prostate cancer cell lines. Morphological examination was performed, as it is regarded as one of the most reliable parameters for the detection of apoptotic changes. Complementarily, biochemical and flow cytometric studies were also used. Characteristical changes of apoptosis were demonstrated in PC-3, Du 145, and LNCaP cancer cells after treatment with etoposide. These cells, thus, retain the ability to undergo apoptosis under adequate conditions, in a promising approach to hormone refractory prostate cancer therapy.     

Substructure in the cell survival response at low radiation dose: effect of different subpopulations

In order to obtain more accurate measurements of cell survival after low doses of radiation, we have used the cell sorter assay, in which a cell sorter is used to accurately count out the number of cells plated for colony formation. This method, combined with data averaging, permits measurements of survival with superior precision, which have revealed that there is substructure in the radiation response of asynchronously dividing Chinese hamster cells. The substructure, observed at doses of a few Gy, has features of a 2-component response, consistent with the presence of subpopulations of cells of different cell-cycle-related radiosensitivity. The absence of any substructure in the radiation response of homogeneous (tightly synchronized) cell populations lends strong support to this subpopulation explanation of the substructure. This assay has also been used on a variety of human tumour cell lines, most of which exhibited substructure similar to that of Chinese hamster cells. This paper outlines the application of the cell sorter assay to three different problems: (i) radiosensitizer mechanisms-etanidazole and RB 6145 are shown to enhance primarily the beta term and alpha term, respectively, of tumour cell kill, indicating that sensitizer efficacy may be tumour-specific and predictable from tumour response parameters; (ii) accurate measurement of Relative Biological Effectiveness (RBE) in a modulated clinical proton beam shows that the RBE is both dose- and depth-dependent; and (iii) measurements at lower doses clearly demonstrate a second order of substructure, termed the hypersensitive response, at doses < 1 Gy.     

Interleukin-10 production by human carcinoma cell lines and its relationship to interleukin-6 expression

Recent data indicate a major role for IL-10 in suppressing immune and inflammatory reactions. To date, expression of human IL-10 has been attributed primarily to helper T lymphocytes, activated monocytes, and neoplastic B cells, and was often found to be associated with IL-6 expression. In this study we sought to determine whether non-hematopoietic human tumor cell lines produce IL-10 and, if so, what is the relationship between IL-10 and IL-6. Using ELISA, we determined IL-10 and IL-6 levels in culture supernatants of 48 cell lines established from carcinomas of the kidney, colon, breast and pancreas, malignant melanomas and neuroblastomas. IL-6 protein was secreted by 28 of the tumor cell lines; IL-10 was measurable in 15 cell lines. IL-6 secretion was maximal and most frequent in renal-cancer cell lines, while IL-10 production was found to be highest and most common among cell lines derived from colon carcinomas. IL-10 in conditioned medium of one of the colon carcinoma cell lines (CCL222) was bio-active, as demonstrated in the mouse MC/9 mast-cell-line assay and in human mixed-lymphocyte reactions. In both assays, IL-10 bio-activity was neutralized by an anti-IL-10 monoclonal antibody. Expression of IL-6 and IL-10 was confirmed by RNA analysis using message amplification by PCR and sequencing of amplified cDNA. LPS, IL-1 alpha, and TNF-alpha strongly enhanced the release of IL-6 by RCC cells, but only marginally affected IL-10 production in colon-carcinoma cells. IL-10 secretion by colon-carcinoma cells was moderately stimulated by IFN-gamma and IL-4. Dexamethasone suppressed the release of IL-6, but had no inhibitory effect on IL-10 secretion. Our results demonstrate that tumor cell lines established from certain types of human carcinomas are capable of expressing and releasing IL-6 and/or IL-10, suggesting a role of these cytokines in solid-tumor development and anti-tumor immunity.     

Distinct P-cadherin expression in cultured normal human keratinocytes and squamous cell carcinoma cell lines

A thermal threshold measurer (TTM) apparatus was developed and tested in 12 dry, nonpregnant, culled cows with the purpose of measuring the thermal nociceptive threshold and of finding the response to morphine sulphate dosages. The cows received a cumulative dose (from 0.00 to 0.40 mg/kg BW) of morphine sulphate through a catheter in the jugular vein. The interval between doses was 20 min, and a nociceptive test was performed 15 min after each injection. The TTM device consisted of a 60 W halogen bulb mounted in a 15 cm PVC tube, with a 0.6 s response time probe attached to its end, connected to a thermocouple. The probe measured the response temperature on the skin over the middle phalanges on the dorsum of the forefoot. The radiating heat stimulus from the bulb was instantaneously terminated with the foot-lift response of the tested animal. The nociceptive response to the 0.00 mg/kg dose was considered the baseline and subsequent measurements were expressed in difference from it. Data were evaluated in a regression analysis using the GLM procedure. A significant elevation (P < 0.0001) in the nociceptive threshold of the cows with cumulative dosing of morphine sulphate was noticed. A high variability (P < 0.0001) in the response among animals was also detected, suggesting that a 2-step dose of morphine sulphate is necessary to achieve a certain degree of induced analgesia in all cows. The nociceptive assay described, using the TTM device, was able to detect an elevation of the thermal threshold of cows due to morphine sulphate induced analgesia. An increase in locomotory behaviour or other side effects due to morphine sulphate were not noticed.     

2-Deoxy-D-glucose preferentially kills multidrug-resistant human KB carcinoma cell lines by apoptosis

The aim of this study was to determine the mechanism of cell death associated with the preferential killing of multidrug-resistant (MDR) cells by the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in a range of MDR human KB carcinoma cell lines selected in different drugs. The D10 values for KB-V1, KB-C1 and KB-A1 (selected in vinblastine, colchicine and doxorubicin respectively) were 1.74, 1.04 and 0.31 mM, respectively, compared with 4.60 mM for the parental cell line (KB-3-1). The mechanism of cell death was identified as apoptosis, based on nuclear morphology, annexin V binding and poly(ADP-ribose) polymerase (PARP) cleavage. 2DG induced apoptosis in the three MDR cell lines in a dose- and time-dependent manner and did not induce necrosis. PARP cleavage was detected in KB-C1 cells within 2 h of exposure to 50 mM 2DG and slightly later in KB-A1 and KB-V1 cells. The relative levels of 2DG sensitivity did not correlate with the levels of multidrug resistance or with the reduced levels of the glucose transporter GLUT-1 in these cells. We speculate that a 2DG-stimulated apoptotic pathway in MDR KB cells differs from that in normal KB cells.