Bystander cell proliferation is modulated by the number of adjacent cells that were exposed to ionizing radiation.

BACKGROUND: Direct cell-to-cell contact appears to be a prerequisite for the proliferative response of bystander WB-F344 cells co-cultured with irradiated cells; however, neither gap junctional intercellular communication nor long-range factors released into the medium appear to be involved (Cytometry 2003;56A:71-80). The present work investigated whether the proliferative bystander response depends on the number of irradiated cells (cells exposed to external gamma-rays or cells exposed to short-range beta-particles emitted by DNA-incorporated (3)H-thymidine) that are adjacent to unirradiated bystander cells. METHODS: Subconfluent monolayers of rat liver epithelial cells (WB-F344) were incubated in the presence of (methyl-(3)H)thymidine at a concentration of 5.8 kBq/ml for 18 h. Radiolabeled cells containing 0.7 x 10(-3) Bq/cell (absorbed dose: 0.14 Gy) were plated together with unlabeled cells in proportions of 6% and 94%, 12% and 88%, 25% and 75%, 50% and 50%, and 75% and 25%, respectively, keeping constant the total number of plated cells. In a parallel experiment, cells acutely exposed to 5 Gy of (137)Cs gamma-rays were plated with unirradiated cells in the same proportions. In both experiments, cells were co-cultured for 24 h followed by a flow cytometric study of their proliferation. The two cell populations in the co-cultures were distinguished by staining one population with carboxyfluorescein diacetate, succinimidyl ester, which metabolizes intracellularly. RESULTS: Increasing the fraction of irradiated cells relative to unirradiated bystander cells led to an increase in proliferation of bystander cells. Specifically, in co-cultures in which irradiated cells were initially mixed with unirradiated cells in proportions of 50% and 50% and of 75% and 25%, respectively, bystander cells showed a statistically significant increase of their proliferation compared with the controls. CONCLUSIONS: The proliferative response of WB-F344 bystander cells is modulated by the number of adjacent cells that are exposed to ionizing radiation from external gamma-rays or intracellularly emitted (3)H beta-particles.     

Radiolabeling of DNA can induce its fragmentation in HL-60 human promyelocytic leukemic cells.

Incorporation of radiolabeled thymidine is commonly used to investigate DNA damage. Using a filter-binding assay, we observed that the addition of various doses of [methyl-3H]thymidine (0.2 and 2 microCi/ml) or [2-14C]thymidine (0.02 and 0.2 microCi/ml) in the culture medium for 2 days, a standard method for cell-labeling, induces DNA fragmentation in HL-60 human promyelocytic cells. This effect was dose- and time-dependent and the DNA fragments were not protein-linked since the levels of DNA fragmentation were identical in the presence and in the absence of proteinase K (0.5 mg/ml). Radiolabeled thymidine-induced DNA fragmentation was associated with an inhibition of cell growth, but cells remained able to exclude trypan blue, suggesting that plasma membrane integrity was conserved, except at very high doses of [methyl-3H]thymidine (2 microCi/ml). By agarose-gel electrophoresis, the DNA-fragmentation was demonstrated to be internucleosomal with a typical ladder pattern. Addition of unlabeled thymidine to the culture medium prevented DNA fragmentation in a dose-dependent manner, indicating that radiolabeled thymidine incorporation in DNA was directly responsible for DNA fragmentation. We conclude that radiolabeling of DNA using thymidine incorporation can induce DNA fragmentation in some cell lines such as HL-60. This observation must be taken into account in methods using radiolabeling to study DNA damage in these cells.     

Free radical-initiated and gap junction-mediated bystander effect due to nonuniform distribution of incorporated radioactivity in a three-dimensional tissue culture model

To investigate the biological effects of nonuniform distribution of radioactivity in mammalian cells, we have developed a novel three-dimensional tissue culture model. Chinese hamster V79 cells were labeled with tritiated thymidine and mixed with unlabeled cells, and multicellular clusters (approximately 1.6 mm in diameter) were formed by gentle centrifugation. The short-range beta particles emitted by (3)H impart only self-irradiation of labeled cells without significant cross-irradiation of unlabeled bystander cells. The clusters were assembled in the absence or presence of 10% dimethyl sulfoxide (DMSO) and/or 100 microM lindane. DMSO is a hydroxyl radical scavenger, whereas lindane is an inhibitor of gap junctional intercellular communication. The clusters were maintained at 10.5 degrees C for 72 h to allow (3)H decays to accumulate and then dismantled, and the cells were plated for colony formation. When 100% of the cells were labeled, the surviving fraction was exponentially dependent on the mean level of radioactivity per labeled cell. A two-component exponential response was observed when either 50 or 10% of the cells were labeled. Though both DMSO and lindane significantly protected the unlabeled or bystander cells when 50 or 10% of the cells were labeled, the effect of lindane was greater than that of DMSO. In both cases, the combined treatment (DMSO + lindane) elicited maximum protection of the bystander cells. These results suggest that the bystander effects caused by nonuniform distributions of radioactivity are affected by the fraction of cells that are labeled. Furthermore, at least a part of these bystander effects are initiated by free radicals and are likely to be mediated by gap junctional intercellular communication.     

Tentative novel mechanism of the bystander effect in glioma gene therapy with HSV-TK/GCV system.

Although many works support gap junctional intercellular communication (GJIC) having a close relation to bystander cell killing in herpes simplex virus thymidine kinase (HSV-TK) gene and ganciclovir (GCV) treatment, our previous work suggested that other factors involved in bystander effect besides GJIC exist. To confirm our primary work, we evaluated the mode of the bystander cell (C6) co-cultured with TK-positive cells (TF10.2) in our designed "insert plates" in which two cell lines could be separated but share the same medium. Another method that we used was adding the supernatant from the medium of GCV-treated TF10.2 cells to the wild type C6. Growth inhibition of the bystander cells was observed despite the absence of GJIC. In addition, apoptotic cell death of TK+ cells and bystander cells was obvious. These studies suggested that other pathways besides cell-cell contacts may play a role in bystander cell killing; the factors released from TK-positive cells could induce apoptosis of bystander cells.     

Glycine-extended gastrin exerts growth-promoting effects on human colon cancer cells.

BACKGROUND: Since human colon cancers often contain significant quantities of progastrin-processing intermediates, we sought to explore the possibility that the biosynthetic precursor of fully processed amidated gastrin, glycine-extended gastrin, may exert trophic effects on human colonic cancer cells. MATERIALS AND METHODS: Binding of radiolabeled glycine-extended and amidated gastrins was assessed on five human cancer cell lines: LoVo, HT 29, HCT 116, Colo 320DM, and T 84. Trophic actions of the peptides were assessed by increases in [3H]thymidine incorporation and cell number. Gastrin expression was determined by northern blot and radioimmunoassay. RESULTS: Amidated gastrin did not bind to or stimulate the growth of any of the five cell lines. In contrast, saturable binding of radiolabeled glycine-extended gastrin was seen on LoVo and HT 29 cells that was not inhibited by amidated gastrin (10(-6) M) nor by a gastrin/CCKB receptor antagonist (PD 134308). Glycine-extended gastrin induced a dose-dependent increase in [3H]thymidine uptake in LoVo (143 +/- 8% versus control at 10(-10) M) and HT 29 (151 +/- 11% versus control at 10(-10) M) cells that was not inhibited by PD 134308 or by a mitogen-activated protein (MAP) or ERK kinase (MEK) inhibitor (PD 98509). Glycine-extended gastrin did stimulate jun-kinase activity in LoVo and HT 29 cells. The two cell lines expressed the gastrin gene at low levels and secreted small amounts of amidated gastrin and glycine-extended gastrin into the media. CONCLUSIONS: Glycine-extended gastrin receptors are present on human colon cancer cells that mediate glycine-extended gastrin's trophic effects via a MEK-independent mechanism. This suggests that glycine-extended gastrin and its novel receptors may play a role in colon cancer cell growth.     

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation

Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.     

Cell surface receptor-antibody association constants and enumeration of receptor sites for monoclonal antibodies

BACKGROUND: Fluorescent markers (labeled antibodies) and flow cytometry are used to enumerate the average number of receptors (antigens) on formed bodies (cells) in whole blood by using a new method that avoids the extra steps of separating bound from unbound fluorescent markers or the use of external standards. METHODS: Mean channel fluorescence intensities of equilibrated marker-cell suspension mixtures, total concentrations of marker, and targeted cell counts obtained by standard cytometry procedures are used to complete the analyses for receptors per cell. Also, flow cytometric assays using competitive binding between fluorescent marker (CD4-RD1, CD8-FITC, CD3-FITC, CD3-RD1) and unlabeled antibody (CD4, CD8, CD3, CD3-dextran) for receptors on white blood cells in whole blood are described for determination of relative and specific binding constants of unlabeled/labeled antibody for targeted receptors. RESULTS: Ranges that were obtained for receptors per cell (lymphocytes) in normal blood donors were as follows: CD4, 4.9 x 10(4)-1.5 x 10(5); CD8, 5.0 x 10(5)-2.1 x 10(6); CD3, 6.6-7.8 x 10(5). Binding constants were highest for unlabeled CD4 antibody, 2. 7 x 10(10)-2.1 x 10(12) M(-1), and then unlabeled CD3 antibody, 1.1 x 10(10)-1.9 x 10(11) M(-1). FITC- and RD1-labeled antibodies typically had binding constants that were 10-to 100-fold lower than the native antibodies. CONCLUSIONS: Values of receptors per cell and binding constants obtained by the new method from flow cytometric analyses of mixtures of whole blood with FITC- or RD1-labeled CD4, CD8, and CD3 antibodies compare well with literature values determined by other methods.     

A bystander effect in alpha-particle irradiations of human prostate tumor cells

Alpha-particle exposures were used to determine whether cells of the human prostate carcinoma cell line DU-145 can produce and respond to a bystander effect signal. An apparatus for alpha-particle irradiation of cells growing as a monolayer on a 1.4-microm-thick Mylar membrane directly above an 241Am alpha-particle source was constructed and calibrated. At the cell irradiation position, the alpha-particle fluence was 998 counts/mm2 s(-1), the average alpha-particle energy was 3.14 MeV, and the average linear energy transfer was 128 keV/microm. The average dose rate to the cells growing on the Mylar surface was 1.2 Gy/min. A co-culture system was used to examine bystander effects transmitted through the medium from the directly targeted cells to tumor cells growing on an insert well beyond the range of the alpha particles. Alpha-particle doses from 0.1 to 6.0 Gy to the targeted cells on the Mylar membrane, followed by a 2-h co-incubation of the cells on the insert in the irradiated medium above the irradiated cells, all caused an approximately 50% increase in micronucleus formation in the nontargeted co-cultured cells. Addition of the radical scavenger DMSO to the medium during the irradiation and the 2-h postirradiation incubation period completely blocked the bystander effect, whereas addition of a nitric oxide scavenger had no effect. Irradiation of medium containing serum, followed by a 2-h incubation, caused no bystander effect in the co-cultured cells. When the co-cultured cells on the insert were placed into the irradiated medium above the directly targeted cells immediately (approximately 1 min) after the irradiation and co-incubated for 2 h, there was no bystander effect. These data indicate that the observed bystander effect requires that the co-cultured cells be present in the medium during the irradiation of the directly targeted cells and suggest the involvement of a short-lived radical species.     

Hydrocortisone in culture protects the blast cells in acute myeloblastic leukemia from the lethal effects of cytosine arabinoside

The blast cells in acute myeloblastic leukemia (AML) respond to many of the same regulatory mechanisms that control normal hemopoiesis. These include the growth factors that bind to membrane receptors and steroid hormones or vitamins that have intracellular receptors. We report the effects in culture of the steroid glucocorticoid hydrocortisone on freshly explanted AML blasts from patients and on two continuous AML cell lines. Only small changes in clonogenic cell numbers in suspension cultures were seen in the presence of hydrocortisone. The most striking effect of the hormone was on the sensitivity of blasts cells to cytosine arabinoside (ara-C). In contrast to the response of AML blast cells to retinoic acid, a ligand for intracellular steroid receptors that sensitizes some blast populations to ara-C, hydrocortisone reduced the toxic effects of the drug. The protective action of hydrocortisone was not mediated through the cell cycle since exposure of blasts to hydrocortisone did not affect the percentage of cells in DNA synthesis as measured with the tritiated thymidine (3HTdR) "suicide" technique. The hydrocortisone effect could be demonstrated using a pulse (20 min) exposure protocol. Blasts pulsed with increasing specific activities of 3HTdR showed the usual response pattern with an initial loss in plating efficiency to about 50% of control, followed by a plateau, regardless of whether the cells had been exposed to hydrocortisone. Control blasts exposed to increasing ara-C concentrations gave very similar dose-response curves; in striking contrast, blast cells cultured in hydrocortisone, then pulsed with ara-C did not lose colony-forming ability even though the same population was sensitive to 3HTdR. The hydrocortisone effect was dose and time related; protection from ara-C increased from 10(-8) to 10(-5) M and was seen after 4 hr exposure but required 8 hr to reach a maximum. We conclude that hydrocortisone can protect blasts from the lethal effects of ara-C even while the cells are in active DNA synthesis.     

Thrombin binds to murine bone marrow-derived macrophages and enhances colony-stimulating factor-1-driven mitogenesis

The binding and mitogenic properties of thrombin have been established in various transformed cell lines. In such systems, thrombin induces cell division in the absence of exogenous growth factors, and the enzyme is considered to act directly as a mitogen. This study explores thrombin's interaction with nontransformed, growth factor-dependent cells. Binding of 125I-alpha-thrombin to colony-stimulating factor (CSF)-1-dependent bone marrow-derived macrophages is saturable, time-dependent, and displaceable by both unlabeled alpha-thrombin, and esterolytically inactive thrombin. Both dissociation studies of pre-bound radio-labeled thrombin and Scatchard analysis assisted by the program "Ligand" suggest adherence of thrombin-binding data to a multi-site model. There are an estimated 2 x 10(4) high affinity sites (Kd = 7 x 10(-9)M) and 2 x 10(6) low affinity sites (Kd = 9 x 10(-7)M) per cell. Quiescent bone marrow-derived macrophages were cultured with either 10(-8)M thrombin, 1000 units of CSF-1/ml, or both and [3H]thymidine incorporation was determined. Thrombin alone did not induce mitogenesis. CSF-1 induced mitogenesis with peak [3H] thymidine incorporation occurring 24 h after addition of the mitogen. This CSF-1-dependent mitogenic influence was enhanced greater than 2-fold by treatment with thrombin.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Crypt base columnar cells in ileum of BDF1 male mice--their numbers and some features of their proliferation

Some features of the proliferative cells at the bottom of the ileal crypts in BDF1 mice have been studied in relation to the distribution of Paneth cells (PC) in an attempt to clarify the nature and function of these crypt base columnar cells (BCC) and to elucidate some aspects of the role of the microenvironment created by the PC. Longitudinal sections of crypts have shown that the ratio of PC to the BCC, which are scattered amongst the PC, is 2.7:1 in sections or approximately 29 PC and 9 BCC per whole crypt, i.e., a ratio of 3.2:1. The labelling index of BCC is about 35%, which is comparable to that of mid-crypt columnar cells. Although the BCC do become labeled, it is concluded that they cannot create vertical pairs or runs of several adjacent BCC since this would seriously disturb the distribution of Paneth cells. Only in dividing crypts are such runs (consisting of 3 to 5 cells) observed. The ability of BCC to synthesize DNA is not dependent on their position in the Paneth cell zone. In 95% of the crypts, the highest Paneth cell is below the 7th cell position from the bottom of the crypt, and the positions of the highest PC on either side of a given crypt are similar. The secreted granules or the cytoplasm of PC specifically bind pokeweed lectin, and this can be used for identification. Tracer doses of 3HTdR (37 kBq/gm body weight) result in the histological death of some BCC, and these damaged cells are evenly distributed throughout the Paneth cell zone. These tracer doses are somewhat selectively incorporated into BCC, i.e., the BCC have a higher grain count in autoradiographs, probably because they possess more thymidine kinase enzyme activity. This ability is very sensitive to the withdrawal of food, because 24 hr of fasting abolished the observed gradient in the intensity of labelling, which is very well correlated with the distribution of BCC. Regeneration of the crypts following cytotoxic exposure to Ara-C is initiated at the base of the crypt and hence may involve the BCC with possible help from the Paneth cells. The latter are insensitive to cytotoxic (S phase specific) agents and may help in the regeneration by preserving the architecture of the base of the crypt.     

Migratory neural stem cells for improved thymidine kinase-based gene therapy of malignant gliomas

Gene therapy of glioma based on viral delivery of herpes simplex virus type I thymidine kinase (HSV-TK) has failed in the clinic because of low transduction efficacy. To circumvent this problem, this study evaluated highly migratory HSV-TK-transduced neural stem cells (NSC) for their ability to kill untransduced glioma cells by a gap junction-mediated bystander effect. The admixture of HSV-TK-transduced NSC to U87MG and LN-18 human malignant glioma cell lines at ratios of 1:10 or 1:1 eliminated more than 50% or 90% of glioma cells in the presence of ganciclovir (25 microM). Glioma cell cytotoxicity required cell-cell contact. Similarly, tumor cell cytotoxicity was observed in two of three primary glioblastoma cell cultures, and the presence of this bystander effect correlated with the expression of connexin 43 in the untransduced glioma target cells. In conclusion, we delineate a role for migratory HSV-transfected NSC to eliminate glioma cells purely by means of the bystander effect.     

Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects

Other investigators have demonstrated by transfer of medium from irradiated cells and by irradiation with low-fluence alpha particles or microbeams that cells do not have to be directly exposed to ionizing radiation to be detrimentally affected, i.e. bystander effects. In this study, we demonstrate by transfer of medium from X-irradiated human CGL1 hybrid cells that the killing of bystander cells reduces the plating efficiency of the nonirradiated CGL1 cells by 33 +/- 6%. In addition, we show that the amount of cell death induced by bystander effects is not dependent on X-ray dose, and that the induction of apoptosis does not appear to be responsible for the cell death. Furthermore, we found that the reduction in plating efficiency in bystander cells is evident for over 18 days, or 22 cell population doublings, after medium transfer, despite repeated refeeding of the cell cultures. Finally, we report the novel observation that bystander effects induced by the transfer of medium from irradiated cells can induce neoplastic transformation. Exposing unirradiated CGL1 cells to medium from cells irradiated with 5 or 7 Gy increased the frequency of neoplastic transformation significantly from 6.3 x 10(-6) in unirradiated controls to 2.3 x 10(-5) (a factor of nearly four). We conclude that the bystander effect induces persistent, long-term, transmissible changes in the progeny of CGL1 cells that result in delayed death and neoplastic transformation. The data suggest that neoplastic transformation in bystander cells may play a significant role in radiation-induced neoplastic transformation at lower doses of X rays.     

Rescue effects in radiobiology: unirradiated bystander cells assist irradiated cells through intercellular signal feedback

Mammalian cells respond to ionization radiation by sending out extracellular signals to affect non-irradiated neighboring cells, which is referred to as radiation induced bystander effect. In the present paper, we described a phenomenon entitled the "rescue effects", where the bystander cells rescued the irradiated cells through intercellular signal feedback. The effect was observed in both human primary fibroblast (NHLF) and cancer cells (HeLa) using two-cell co-culture systems. After co-culturing irradiated cells with unirradiated bystander cells for 24h, the numbers of 53BP1 foci, corresponding to the number of DNA double-strand breaks in the irradiated cells were less than those in the irradiated cells that were not co-cultured with the bystander cells (0.78±0.04foci/cell vs. 0.90±0.04foci/cell) at a statistically significant level. Similarly, both micronucleus formation and extent of apoptosis in the irradiated cells were different at statistically significant levels if they were co-cultured with the bystander cells. Furthermore, it was found that unirradiated normal cells would also reduce the micronucleus formation in irradiated cancer cells. These results suggested that the rescue effects could participate in repairing the radiation-induced DNA damages through a media-mediated signaling feedback, thereby mitigating the cytotoxicity and genotoxicity of ionizing radiation.     

Radiation-induced bystander effects in cultured human stem cells

Background

The radiation-induced “bystander effect” (RIBE) was shown to occur in a number of experimental systems both in vitro and in vivo as a result of exposure to ionizing radiation (IR). RIBE manifests itself by intercellular communication from irradiated cells to non-irradiated cells which may cause DNA damage and eventual death in these bystander cells. It is known that human stem cells (hSC) are ultimately involved in numerous crucial biological processes such as embryologic development; maintenance of normal homeostasis; aging; and aging-related pathologies such as cancerogenesis and other diseases. However, very little is known about radiation-induced bystander effect in hSC. To mechanistically interrogate RIBE responses and to gain novel insights into RIBE specifically in hSC compartment, both medium transfer and cell co-culture bystander protocols were employed.

Methodology/Principal Findings

Human bone-marrow mesenchymal stem cells (hMSC) and embryonic stem cells (hESC) were irradiated with doses 0.2 Gy, 2 Gy and 10 Gy of X-rays, allowed to recover either for 1 hr or 24 hr. Then conditioned medium was collected and transferred to non-irradiated hSC for time course studies. In addition, irradiated hMSC were labeled with a vital CMRA dye and co-cultured with non-irradiated bystander hMSC. The medium transfer data showed no evidence for RIBE either in hMSC and hESC by the criteria of induction of DNA damage and for apoptotic cell death compared to non-irradiated cells (p>0.05). A lack of robust RIBE was also demonstrated in hMSC co-cultured with irradiated cells (p>0.05).

Conclusions/Significance

These data indicate that hSC might not be susceptible to damaging effects of RIBE signaling compared to differentiated adult human somatic cells as shown previously. This finding could have profound implications in a field of radiation biology/oncology, in evaluating radiation risk of IR exposures, and for the safety and efficacy of hSC regenerative-based therapies.     

Inhibitory and stimulatory bystander effects are differentially induced by Iodine-125 and Iodine-123

The bystander effect, originating from cells irradiated in vitro, describes responses of surrounding cells not targeted by the radiation. Previously we demonstrated that the subcutaneous injection into nude mice of human adenocarcinoma LS174T cells lethally irradiated by Auger electrons from the decay of DNA-incorporated (125)I inhibits growth of co-injected LS174T cells (inhibitory bystander effect; Proc. Natl. Acad. Sci. USA 99, 13765-13770, 2002). We have repeated these studies using cells exposed to lethal doses of (123)I, an Auger electron emitter whose emission spectrum is identical to that of (125)I, and report herein that the decay of (123)I within tumor cell DNA stimulates the proliferation of neighboring unlabeled tumor cells growing subcutaneously in nude mice (stimulatory bystander effect). Similar inhibitory bystander effects ((125)I) and stimulatory bystander effects ((123)I) are obtained in vitro. Moreover, supernatants from cultures with (125)I-labeled cells are positive for tissue inhibitors of metalloproteinases (TIMP1 and TIMP2), and those from cultures with (123)I-labeled cells are positive for angiogenin. These findings call for the re-evaluation of current dosimetric approaches for the estimation of dose-response relationships in individuals after radiopharmaceutical administration or radiocontamination and demonstrate a need to adjust all "calculated" dose estimates by a dose modification factor (DMF), a radionuclide-specific constant that factors in hitherto not-so-well recognized biophysical processes.     

Binding of phosphatidylethanolamine and phosphatidylinositol to OKT8+ lymphocytes activates suppressor cell activity

The phospholipids phosphatidylethanolamine (PE) and phosphatidylinositol (PI) have been shown to activate a population of OKT8-enriched lymphocytes to become activated suppressor cells that result in the suppression of lymphocyte blastogenesis to a variety of mitogens and antigens. This suppression is dose dependent, and maximal suppressor activity is obtained at concentrations of 125 micrograms/ml PE and 25 micrograms/ml PI. Activation of the suppressor cell population is not associated with an actual increase in the number of cells expressing the OKT8 antigen, but the number of these cells expressing Dr antigens on their surface was increased. Both PE and PI bound to lymphocytes in a specific manner. Binding of radiolabeled PE could be inhibited by unlabeled PE but not by PI or phosphatidylserine (PS). Similarly, the binding of PI to lymphocytes was also found to be specific. Although radiolabeled PE bound to lymphocytes other than OKT8+ cells, and to other peripheral leukocytes, it bound to OKT8+ cells with a significantly greater affinity than it did to the other cell types. The Kd for PE was 1 x 10(2) nM and for PI was 1 x 10(3) nM, and receptor cell densities for these two phospholipids were estimated at 1 x 10(-8) nM and 3 x 10(-9) nM, respectively. The receptors for these two phospholipids were trypsin and heat sensitive, and the receptor sites could be regenerated after a 24-hr incubation after trypsinization.     

Somatic mutagenesis at T-cell receptor locus in inhabitants of radiation polluted regions as a result of the Chernobyl disaster

In the period of 2001-2004, frequency of cells bearing mutations at T-cell receptor (TCR) locus was assessed in 553 inhabitants of radiation polluted regions of the Russian Federation and 154 unexposed control persons. The inhabitants were divided into three groups according to age at the moment of the Chernobyl disaster and 137Cs pollution density: 1) in utero, 37-555 kBq/m2; 2) 0-14 years old, 20-555 kBq/m2; 3) 18 and more years old, highest 137Cs density (185 more than 555 kBq/m2). The most intense changes of the TCR-mutant cell frequency were observed in the group of persons exposed to ionizing radiation in utero. The mean frequency of the mutant cells was higher in the first group than in age-matched control group by about 1.5-fold: 4.0 x 10(-4) vs 2.7 x 10(-4) accordingly (p < 0.0001). Elevation in the mean TCR-mutant cell frequency was less expressed in group of inhabitants aged 0-14 years at the moment of irradiation start: 1.3-fold increase in comparison to age-matched control (3.8 x 10(-4) vs 2.9 x 10(-4), p = 0.0002). It was not found significant differences in mutant cell frequencies between control group and adults consisting in the third group (18 and more years old at the moment of the Chernobyl accident). The changes of the TCR-mutant cell frequency in persons exposed in pre- and postnatal periods differ not only quantitatively, but qualitatively. In the fist case all persons react to irradiation by increasing number of the TCR-mutant cells in some degree. In the second case - only a part of population. Proportion of reacting persons depends on age at the start of irradiation and, perhaps, on dose absorbed. The TCR-mutant frequency was significantly higher in persons with benign tumors of different localizations and nodules in thyroid gland than in persons without this pathology.     

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions.

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.