hSMG-1 and ATM sequentially and independently regulate the G1 checkpoint during oxidative stress.

Genotoxic stress activates the phosphatidylinositol 3-kinase-like kinases (PIKKs) that phosphorylate proteins involved in cell cycle arrest, DNA repair and apoptosis. Previous work showed that the PIKK ataxia telangiectasia mutated (ATM) but not ATM and Rad3 related phosphorylates p53 (Ser15) during hyperoxia, a model of prolonged oxidative stress and DNA damage. Here, we show hSMG-1 is responsible for the rapid and early phosphorylation of p53 (Ser15) and that ATM helps maintain phosphorylation after 24 h. Despite reduced p53 phosphorylation and abundance in cells depleted of hSMG-1 or ATM, levels of the p53 target p21 were still elevated and the G(1) checkpoint remained intact. Conditional overexpression of p21 in p53-deficient cells revealed that hyperoxia also stimulates wortmannin-sensitive degradation of p21. siRNA depletion of hSMG-1 or ATM restored p21 stability and the G(1) checkpoint during hyperoxia. These findings establish hSMG-1 as a proximal regulator of DNA damage signaling and reveal that the G(1) checkpoint is tightly regulated during prolonged oxidative stress by both PIKK-dependent synthesis and proteolysis of p21.

Effects of p53 mutations on cellular sensitivity to ionizing radiation.

Mutations in the p53 tumor suppressor gene have been found in more than 50% of human tumors including those in breast, colon, lung, and oral cavity. However, the significance of p53 mutation in radiation sensitivity and its underlying mechanisms still remains unclear. In this study, we have measured the effects of p53 mutation on cell cycle delay, apoptosis, and radiation sensitivity using mouse cells transfected with different forms of p53 mutations. Wild-type p53 and p53-Null mouse embryo fibroblast cells were used as positive and negative controls, respectively. Exponentially growing cells were irradiated with 0- to 9-Gy gamma rays and then assayed for cell survival, p53 expression, cell cycle checkpoint, and apoptosis. Cell survivals determined by clonogenic assay show that p53 mutant cells are generally more sensitive to ionizing radiation than cells with wild-type p53. Western blot analysis indicates that exposure to 6-Gy gamma rays increases the p53 expression levels by two- to threefold in wild-type p53 cells. However, the p53 level remains unchanged in cells with mutant p53 during the same postirradiation period. Irradiation with 6-Gy gamma rays produces G2/M arrest in all cell lines, indicating that p53 is probably not involved in the G2/M checkpoint. However, all mutant cells fail to show any significant G1/S arrest after irradiation, suggesting that G1/S arrest may be implicated in radiation sensitivity. Finally, there is very little apoptosis (<3% by Tat-mediated dUTP nick-end labeling [TUNNEL] and morphologic assays) detected in wild-type and p53 mutant cell lines after 6-Gy gamma rays. Our results suggest that mutant forms of p53 represent a phenotype that affects the radiation sensitivity and is not dependent on the apoptotic pathway.

The cyclin-dependent kinase inhibitor p21 protects the lung from oxidative stress.

The lung is a major target tissue for oxidative stress, including hyperoxia used to relieve tissue hypoxia. Unfortunately, severe hyperoxia damages DNA, inhibits proliferation, and kills cells, resulting in morbidity and mortality. Although hyperoxia induces the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(Cip1/WAF1/Sdi1) (p21), their role in pulmonary injury remains unknown. Using p53- and p21-deficient mice we demonstrate that hyperoxia induces p21 in the absence of p53, suggesting that previous conclusions that p53 does not modify hyperoxic lung injury cannot be extrapolated to p21. In fact, mean survival of p21-deficient mice decreased by 40% and was associated with terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling staining of alveolar debris, indicative of DNA fragmentation and cell death. Ultrastructural analyses revealed that alveolar endothelial and type I epithelial cells died rapidly by necrosis. Although hyperoxia decreased DNA replication in p21-wild-type lungs, it had no effect on replication in p21-deficient lungs. Our findings suggest that p21 protects the lung from oxidative stress, in part, by inhibiting DNA replication and thereby allowing additional time to repair damaged DNA. Our findings have implications for patients suffering from the toxic effects of supplemental oxygen therapies.

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia.

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.

Sigma-2 receptors as a biomarker of proliferation in solid tumours.

Over the past several years, our group has provided considerable evidence that the expression of sigma-2 (sigma2) receptors may serve as a biomarker of tumour cell proliferation. In these in vitro studies, sigma2 receptors were expressed 8-10 times more in proliferative (P) tumour cells than in quiescent (Q) tumour cells, and the extent and kinetics of their expression were independent of a number of biological, physiological and environmental factors often found in solid tumours. Moreover, the expression of sigma2 receptors followed both the population growth kinetics when Q-cells were recruited into the P-cell compartment and the proliferative status of human breast tumour cells treated with cytostatic concentrations of tamoxifen. However, these in vitro studies may or may not be indicative of what might occur in solid tumours. In the present study, the sigma2 receptor P:Q ratio was determined for the cells from subcutaneous 66 (diploid) and 67 (aneuploid) tumours grown in female nude mice. The sigma2 receptor P:Q ratio of the 66 tumours was 10.6 compared to the sigma2 receptor P:Q ratio of 9.5 measured for the 66 tissue culture model. The sigma2 receptor P:Q ratio of the 67 tumours was 4.5 compared to the sigma2 receptor P:Q ratio of approximately equal 8 measured for the 67 tissue culture model. The agreement between the solid tumour and tissue culture data indicates that: (1) the expression of sigma2 receptors may be a reliable biomarker of the proliferative status of solid tumours and (2) radioligands with both high affinity and high selectivity for sigma2 receptors may have the potential to non-invasively assess the proliferative status of human solid tumours using imaging techniques such as positron emission tomography or single-photon emission computerized tomography.

Alteration of tumour response to radiation by interleukin-2 gene transfer.

We have previously shown that BALB/c-derived EMT6 mammary tumours transfected with interleukin (IL)-2 have decreased hypoxia compared to parental tumours, due to increased vascularization. Since hypoxia is a critical factor in the response of tumours to radiation treatment, we compared the radiation response of IL-2-transfected tumours to that of parental EMT6 tumours. Because the IL-2 tumours have an altered host cell composition, which could affect the interpretation of radiation sensitivity as measured by clonogenic cells, we employed flow cytometric analysis to determine the proportion of tumour cells vs host cells in each tumour type. Using this approach, we were able to correct the plating efficiency based on the number of actual tumour cells derived from tumours, making the comparison of the two types of tumours possible. We also excluded the possibility that cytotoxic T-cells present in EMT6/IL-2 tumours could influence the outcome of the clonogenic cell survival assay, by demonstrating that the plating efficiency of cells derived from EMT6/IL-2 tumours remained unchanged after depletion of Thy-1+ cells. The in vivo radiation response results demonstrated that IL-2-transfected tumours were more sensitive to radiation than parental EMT6 tumours. The hypoxic fraction of the EMT6/IL-2 tumours growing in vivo was markedly decreased relative to parental EMT6 tumours thus the increased sensitivity results from the increased vascularity we have previously observed in these tumours. These results indicate the potential therapeutic benefit of combining radiation and immunotherapy in the treatment of tumours.

p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia.

Previous studies have shown that lungs of adult mice exposed to >95% oxygen have increased terminal deoxyribonucleotidyltransferase dUTP nick end-label staining and accumulate p53, the expression of which increases in cells exposed to DNA-damaging agents. The present study was designed to determine whether hyperoxia also increased expression of the growth arrest and DNA damage (GADD) gene 45 and GADD153, which are induced by genotoxic stress through p53-dependent and -independent pathways. GADD proteins have been shown to inhibit proliferation and stimulate DNA repair and/or apoptosis. GADD45 and GADD153 mRNAs were not detected in lungs exposed to room air but were detected after 48 and 72 h of exposure to hyperoxia. In situ hybridization and immunohistochemistry revealed that hyperoxia increased GADD45 and GADD153 expression in the bronchiolar epithelium and GADD45 expression predominantly in alveolar cells that were morphologically consistent with type II cells. Hyperoxia also increased GADD expression in p53-deficient mice. Terminal deoxyribonucleotidyltransferase dUTP nick end-label staining of lung cells from p53 wild-type and p53-null mice exposed to hyperoxia for 48 h revealed that hyperoxia-induced DNA fragmentation was not modified by p53 deficiency. These studies are consistent with the hypothesis that hyperoxia-induced DNA fragmentation is associated with the expression of GADD genes that may participate in DNA repair and/or apoptosis.

Hyperoxia inhibits proliferation of Mv1Lu epithelial cells independent of TGF-beta signaling.

High concentrations of O(2) inhibit epithelial cell proliferation that resumes on recovery in room air. To determine whether growth arrest is mediated by transforming growth factor-beta (TGF-beta), changes in cell proliferation during exposure to hyperoxia were assessed in the mink lung epithelial cell line Mv1Lu and the clonal variant R1B, which is deficient for the type I TGF-beta receptor. Mv1Lu cells treated with TGF-beta accumulated in the G(1) phase of the cell cycle as determined by propidium iodide staining, whereas proliferation of R1B cells was unaffected by TGF-beta. In contrast, hyperoxia inhibited proliferation of both cell lines within 24 h of exposure through an accumulation in the S phase. Mv1Lu cells treated with TGF-beta and exposed to hyperoxia accumulated in the G(1) phase, suggesting that TGF-beta can inhibit the S phase accumulation observed with hyperoxia alone. Cyclin A was detected in cultures exposed to room air or growth arrested by hyperoxia while decreasing in cells growth arrested in the G(1) phase by TGF-beta. Finally, hyperoxia failed to activate a TGF-beta-dependent transcriptional reporter in both Mv1Lu and R1B cells. These findings reveal that simple growth arrest by hyperoxia involves a defect in S phase progression that is independent of TGF-beta signaling.

Effect of ploidy, recruitment, environmental factors, and tamoxifen treatment on the expression of sigma-2 receptors in proliferating and quiescent tumour cells.

Recently, we demonstrated that sigma-2 receptors may have the potential to be a biomarker of tumour cell proliferation (Mach et al (1997) Cancer Res 57: 156-161). If sigma-2 receptors were a biomarker of tumour cell proliferation, they would be amenable to detection by non-invasive imaging procedures, thus eliminating many of the problems associated with the flow cytometric measures of tumour cell proliferation presently used in the clinic. To be a good biomarker of tumour cell proliferation, the expression of sigma-2 receptors must be essentially independent of many of the biological, physiological, and/or environmental properties that are found in solid tumours. In the investigation reported here, the mouse mammary adenocarcinoma lines, 66 (diploid) and 67 (aneuploid), 9L rat brain tumour cells, and MCF-7 human breast tumour cells were used to study the extent and kinetics of expression of sigma-2 receptors in proliferative (P) and quiescent (Q) tumour cells as a function of species, cell type, ploidy, pH, nutrient depletion, metabolic state, recruitment from the Q-cell compartment to the P-cell compartment, and treatment with tamoxifen. In these experiments, the expression of sigma-2 receptors solely reflected the proliferative status of the tumour cells. None of the biological, physiological, or environmental properties that were investigated had a measurable effect on the expression of sigma-2 receptors in these model systems. Consequently, these data suggest that the proliferative status of tumours and normal tissues can be non-invasively assessed using radiolabelled ligands that selectively bind sigma-2 receptors.

Hypoxia significantly reduces aminolaevulinic acid-induced protoporphyrin IX synthesis in EMT6 cells.

We have studied the effects of hypoxia on aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) synthesis in EMT6 monolayer cultures characterized by different cell densities and proliferation rates. Specifically, after ALA incubation under hypoxic or normoxic conditions, we detected spectrofluorometrically the PpIX content of the following populations: (a) low-density exponentially growing cells; (b) high-density fed-plateau cells; and (c) high-density unfed-plateau cells. These populations were selected either for the purpose of comparison with other in vitro studies (low-density exponentially growing cells) or as representatives of tumour regions adjacent to (high-density fed-plateau cells) and further away from (high-density unfed-plateau cells) capillaries. The amount of PpIX per cell produced by each one of these populations was higher after normoxic ALA incubation. The magnitude of the effect of hypoxia on PpIX synthesis was dependent on cell density and proliferation rate. A 42-fold decrease in PpIX fluorescence was observed for the high-density unfed-plateau cells. PpIX production by the low-density exponential cells was affected the least by ALA incubation under hypoxic conditions (1.4-fold decrease), whereas the effect on the high-density fed-plateau population was intermediate (20-fold decrease).

Measurement of proliferation activities in human tumor models: a comparison of flow cytometric methods.

Proliferating cells in tumors may be of considerable relevance in cancer therapy. Not only do such cells dictate the rate of tumor progression, but evidence exists that they may also play an important role in the diagnosis and prognosis of tumor regrowth. Consequently, the identification of this subset of cells in the overall neoplastic cell population is of considerable importance. The aim of the present investigations was to compare four flow cytometric methodologies commonly used to study cell proliferation. These included nuclear antigen Ki67 detection, acridine orange (AO) and bromodeoxyuridine (BrdUrd) staining, and percent S-phase determinations. Three human tumor cell lines (HEp3, A549, H226) were examined in various stages of growth. Further, a direct comparison was made of the proliferation activities of HEp3 cells grown in culture or as xenografts in nude mice. The results showed that of the techniques investigated, detection of the nuclear antigen Ki67 may be most useful for marking proliferating tumor cells and determining tumor growth fractions.

Passive mechanical behavior of human neutrophils: effects of colchicine and paclitaxel.

The role of microtubules in determining the mechanical rigidity of neutrophils was assessed. Neutrophils were treated with colchicine to disrupt microtubules, or with paclitaxel to promote formation of microtubules. Paclitaxel caused an increase in the number of microtubules in the cells as assessed by immunofluorescence, but it had no effect on the presence or organization of actin filaments or on cellular mechanical properties. Colchicine at concentrations <1.0 microM caused disruption of microtubular structures, but had little effect on either F-actin or on cellular mechanical properties. Higher concentrations of colchicine disrupted microtubular structure, but also caused increased actin polymerization and increases in cell rigidity. Treatment with 10 microM colchicine increased F-actin content by 17%, the characteristic cellular viscosity by 30%, the dependence of viscosity on shear rate by 10%, and the cortical tension by 18%. At 100 microM colchicine the corresponding increases were F-actin, 25%; characteristic viscosity, 50%; dependence of viscosity on shear rate, 20%; and cortical tension, 21%. These results indicate that microtubules have little influence on the mechanical properties of neutrophils, and that increases in cellular rigidity caused by high concentrations of colchicine are due to a secondary effect that triggers actin polymerization. This study supports the conclusion that actin filaments are the primary structural determinants of neutrophil mechanical properties.

Toxicity of cadmium in human trophoblast cells (JAr choriocarcinoma): role of calmodulin and the calmodulin inhibitor, zaldaride maleate.

Cadmium (Cd), the heavy metal, is toxic to the placenta. The objectives of this study were to determine if Cd toxicity is due to inhibition of placental or trophoblast cell proliferation through interactions with the intracellular calcium binding protein, calmodulin (CaM). Cd can replace calcium and thus interfere with CaM's function. Also, CaM inhibitors reverse selected toxic effects of Cd. The CaM inhibitor, zaldaride maleate, was used to determine if Cd inhibits trophoblast cell proliferation through interactions with CaM. JAr choriocarcinoma cells, a neoplastic trophoblast cell line which is similar to early human trophoblast cells, were selected to study this question. Cd (20 and 40 microM) inhibits JAr cell proliferation, as measured by cell number and BrdU incorporation. Zaldaride (10 and 20 microM) inhibits proliferation to a lesser extent; 100 microM is lethal. To determine if zaldaride alters actions of Cd, zaldaride and Cd are added simultaneously. Zaldaride (20 microM) and Cd (20 microM) together inhibit proliferation less than Cd alone, thus partially protecting cells. Metallothionein is induced in cells exposed to Cd, while zaldaride does not cause induction of this cellular defense mechanism protein. To determine if Cd inhibits proliferation through alterations of cell cycle, JAr cells enriched for G0/G1 phase were exposed to 20 microM Cd, 20 microM zaldaride, or 20 microM Cd plus 20 microM zaldaride for 24 hr. Cells remain in G0/G1 following Cd exposure; cells treated with 20 microM zaldaride progress through S phase and into G2. Zaldaride and Cd together allow JAr cells to leave G1 and enter S phase, partially relieving the cycle block produced by Cd. This study demonstrates a role for calmodulin in mediating the toxicity of Cd in trophoblast cell proliferation.

Effect of caffeine on gamma-ray induced G2 arrest in well-synchronized Chinese hamster ovary cells in vitro.

G1-rich cells were separated from exponentially growing asynchronous cultured Chinese hamster ovary (CHO-K1) cells by centrifugal elutriation and a Coulter Counter. The G1-rich cells were incubated in medium that contained hydroxyurea (HU) to kill S phase cells and obtain the purest G1/S boundary cells possible. The HU-treated cells were washed, and were again incubated, in medium without HU, to allow these well-synchronized G1/S boundary cells to progress to S and G2/M phases. At various times after release from G1/S boundary, 4 Gy of gamma-ray and/or caffeine was administered to the cells. Eight hours after the removal of HU, cell-cycle analysis was performed with a flow cytometer. G2 arrest induced by gamma-rays was clearly shown when radiation was given earlier than 6.5 hours after HU removal. G2 arrest induced by radiation given 0.5-6.5 hours after HU removal was reduced by caffeine treatment given 6.0-6.5 hours after HU removal. Caffeine released radiation-induced G2 arrest when the radiation was given before the cultured cells entered G2/M phase and when caffeine was applied to the irradiated cells at the time when G1/S boundary cells not treated by radiation or with caffeine entered G2/M phase. Our method of centrifugal elutriation combined with incubation with HU was useful for isolating pure G1/S boundary cells from in vitro asynchronous cultures.

The effect of bcr-abl antisense oligonucleotide on DNA synthesis and apoptosis in K562 chronic myeloid leukemia cells.

Mutations in oncogenes have traditionally been viewed as inducing malignancy by causing excessive cell division. However, an additional possible tumorigenic mechanism is inhibition of normally occurring apoptosis. We have studied the mechanism of action of bcr-abl in chronic myeloid leukemia (CML) by inhibiting its expression using antisense oligonucleotides. K562 cells, derived initially from a patient with CML, were incubated with 16 microM 3',5'-capped bcr-abl antisense phosphodiester 18mer targeting the bcr-abl junctional sequence. Antisense reduced cell number by day 5 by 44% +/- 2.5% S.E. compared to nonsense or no-oligomer controls. Compared to nonsense oligomer, antisense oligomer reduced [3H]thymidine incorporation by only 13% +/- 1%. By the more reliable bromodeoxyuridine incorporation method, antisense had no inhibiting effect on DNA synthesis. In contrast to its minimal effect on DNA synthesis, antisense had a large effect on apoptosis. At day 4, after 3 days of oligomer treatment, antisense increased the proportion of cells with less than 2 N DNA 2.5 +/- 0.3-fold compared to nonsense, as revealed by analysis of DNA distribution following propidium iodide-staining. After 3 days of oligomer treatment and 24 h of serum deprivation, antisense increased the proportion of cells with less than 2 N DNA even more, over 3.1 +/- 1.1-fold compared to nonsense. Because CML cells are resistant to the induction of apoptosis (as judged by DNA laddering on electrophoresis, which requires double-stranded breaks), we also assayed the binding of terminal deoxynucleotidyl transferase (TdT), which requires only single-stranded DNA breaks. Antisense treatment for 3 days increased TdT binding at day 4 by 16.4 +/- 8.7-fold. We conclude that, in CML, bcr-abl may lead to the accumulation of myeloid cells to a greater extent by inhibiting apoptosis than by increasing cell division. This bcr-abl induced inhibition of apoptosis may thwart chemotherapy and foster the accumulation of further mutations leading to the development of the blastic phase of the disease.

Cell cycle-dependence of HL-60 cell deformability.

In this study, the role of cytoskeleton in HL-60 deformability during the cell cycle was investigated. G1, S, and G2/M cell fractions were separated by centrifugal elutriation. Cell deformability was evaluated by pipette aspiration. Tested at the same aspiration pressures, S cells were found to be less deformable than G1 cells. Moreover, HL-60 cells exhibited power-law fluid behavior: mu = mu c(gamma m/ gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate, mu c is the characteristic viscosity at the characteristic shear rate gamma c, and b is a material constant. At a given shear rate, S cells (mu c = 276 +/- 14 Pa.s, b = 0.51 +/- 0.03) were more viscous than G1 cells (mu c = 197 +/- 25, b = 0.53 +/- 0.02). To evaluate the relative importance of different cytoskeletal components in these cell cycle-dependent properties, HL-60 cells were treated with 30 microM dihydrocytochalasin B (DHB) to disrupt F-actin or 100 microM colchicine to collapse microtubules. DHB dramatically softened both G1 and S cells, which reduced the material constants mu c by approximately 65% and b by 20-30%. Colchicine had a limited effect on G1 cells but significantly reduced mu c of S cells (approximately 25%). Thus, F-actin plays the predominate role in determining cell mechanical properties, but disruption of microtubules may also influence the behavior of proliferating cells in a cell cycle-dependent fashion.

Changes in HL-60 cell deformability during differentiation induced by DMSO.

We have investigated changes in cellular deformability during promyelocytic leukemic HL-60 cell maturation. HL-60 cells were induced to mature with 1.25% dimethyl sulfoxide. Cellular deformability was evaluated by single-cell micropipette aspiration at one day, four days and seven days after induction. HL-60 cells were found to decrease in size and increase in deformability with maturation. When tested under the same aspiration pressures (0.5-1.3 kPa), cytoplasmic viscosity was found to vary from 210 to 85 Pa.s for cells prior to induction; it varied from 85 to 40 Pa.s for cells seven days after induction. Further, cytoplasmic viscosity exhibits power-law dependence on shear rate, mu = mu c (gamma m/gamma c)-b, where mu is cytoplasmic viscosity, gamma m is mean shear rate during cell entry, mu c is the characteristic viscosity at the characteristic shear rate, gamma c, and b is a material coefficient. Cells of all maturities showed similar dependence on shear rate (b approximately 0.5), but the characteristic viscosity decreased with maturation except for Day 1. When gamma c was set to 1 s-1, mu c = 236 +/- 5 Pa.s for cells prior to induction, mu c = 239 +/- 7, 209 +/- 7 and 175 +/- 14 Pa.s for cells on Days 1, 4 and 7 of induction, respectively.

Absence of radiation-induced G1 arrest in two closely related human lymphoblast cell lines that differ in p53 status.

In order to examine more precisely the role of p53 in the activation of the G1/S checkpoint by ionizing radiation, we examined two human lymphoblast cell lines derived from the same donor. The TK6 line had a doubling time of 12.2 h and expressed wild type p53, while the WTK1 line had a doubling time of 12.7 h and expressed mutant p53. The two lines differ significantly in their susceptibility to radiation-induced cell killing and apoptosis. Cells were examined by flow cytometry at regular intervals from 0 to 12 h after irradiation with two different doses designed to yield equivalent survival levels in both cell lines. In some experiments, cells were incubated with colcemid to block them in the first postirradiation mitosis and prevent contamination of the flow cytometric profiles with second cycle cells. There was no significant difference between the two cell lines in the progression of irradiated cells out of G1 and into the S and G2 phases of the cell cycle. In particular, there was no evidence for a prolonged arrest in G1 in the TK6 cell line expressing wild type p53. Furthermore, expression of the p53 downstream genes WAF1/CIP1 and RB appeared normal in TK6 cells. These results suggest that factors other than those in the p53 signal transduction pathway alone may be required to activate the G1/S checkpoint in irradiated human cells and that apoptosis and G1 arrest may utilize different pathways.

In vitro radiation sensitivity of mouse lung fibroblasts isolated by flow cytometry.

PURPOSE: Recently, we have isolated two major fibroblast cells (Thy-1+, Thy-1-) from mouse LAF1 lung tissue using the anti-Thy-1 antibody expression and fluorescence activated cell sorter. To examine the possibility that x- or gamma-ray-induced pulmonary fibrosis at the late stage of injury could arise from radioresistant cell subpopulations, the radiation sensitivities of Thy-1+ and Thy-1- cells were evaluated by the colony forming assay. METHODS AND MATERIALS: Cell survival curves, repair of potentially lethal damage (PLD) and sublethal damage (SLD), and cell-age response curves were obtained after Cs-137 gamma-ray irradiation. RESULTS: The cell survival curves measured after 0-10 Gy gamma-ray showed that Thy-1+ cells were slightly more radioresistant than Thy-1- cells. The D0, n, alpha, and beta values measured from the survival curves also confirmed this observation. After a single dose of 10 Gy, a small amount of PLD repair was observed in Thy-1- cells, while no PLD repair was found in Thy-1+ cells. Although the initial cell survival level of Thy-1- cells was lower, the final survival levels of Thy-1+ and Thy-1- cells became identical at 8 h after irradiation due to the PLD repair. After split-dose irradiation of 4 Gy followed by 4 Gy, a similar extent and rate of SLD repair was found in Thy-1+ and Thy-1- cells. Cell-age response curves were obtained from irradiated G0/G1, S, and G2M cells separated by centrifugal elutriation and irradiated with 8 Gy gamma-ray. The results indicated that Thy-1+ and Thy-1- cells had a similar S resistant, and G1, G2M-sensitive radiation cell-age response curve. CONCLUSIONS: This study suggests that the selection of radioresistant lung fibroblast may not be responsible for the development of lung fibrosis in irradiated LAF1 mouse.

Effects of GM-CSF on Ki67 expression and cell cycle traverse in acute myelogenous leukemia specimens and cell lines.

A new strategy in the treatment of acute myelogenous leukemia is to attempt to increase the growth fraction of clonal leukemic cells prior to administration of chemotherapeutic agents by the administration of hematopoietic growth factors. We have studied the effect of GM-CSF on the cell cycle status and Ki67 nuclear antigen expression of AML blasts in vitro. The effect of growth factors and stromal cell co-culture on Ki67 expression in leukemic cell lines was also examined. Neither stromal cell co-culture nor exposure of factor-dependent and factor-independent cell lines to GM-CSF, IL-3, SCF, or combinations thereof significantly changed the percentage of cells expressing Ki67. In the AML population analyzed as a whole, exposure of blasts to GM-CSF for up to 96 h did not significantly change the percentage of cells in S phase or expressing Ki67.