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The molecular mechanism(s) behind the development of endocrine resistance in breast cancer remains controversial. Here, we compare the capability of oestrogen receptor (ER)-negative cells (MDA-231) versus ER-positive tamoxifen-sensitive cells (MCF-7) to handle DNA repair, transmit signals from damaged DNA, initiate cell death via apoptosis, and then to control transmitted signals from the cell cycle and to synthesize growth factors and receptors. Genes related to these events were studied by cDNA micro-array. Normal human breast cells (H2F) and human lymphoblastoid tumour cells (CEM) were used as controls. Of the 18 genes investigated, 10 genes showed differences in their expression between the cell types. The ER-negative cells showed higher expressions of BRCA1, BRCA2, cdc2, cyclin B1, cyclin D1, cyclin E, IGFBP-3, TGF-alpha, TGF beta 2 and a lower expression of TGF beta R1. No differences in the expressions of bax, bcl-2, p53, p21 and GADD45 were found between the two cell lines. We found that the ER-negative cells were characterized by: (1) a stimulated expression of growth factors and cell cycle regulation compounds, (2) improved DNA repair capacity, but (3) no change in DNA damage signals and apoptotic pathways. Improved DNA repair capacity of ER-negative cells would have a growth advantage over ER-positive tumours when receiving antitumour therapy., (Copyright 2004 S. Karger AG, Basel)

A study was made evaluating the use of radiation-induced cell cycle delay in lymphocytes to predict tumour response to radiotherapy. Peripheral blood lymphocytes were isolated from whole blood from 49 patients with head and neck cancer before treatment with radiotherapy and from 25 healthy donors. The clinical response to radiotherapy was assessed at 0-2 months after treatment. The level of radiation-induced cell cycle delay was measured using flow cytometry after mitogen stimulation of lymphocytes. The analysis of ten normal donors gave no significant difference in variability between the intra-assay and the intra-donor samples. However, the cell cycle data for lymphocytes from these healthy donors showed significant inter-individual differences in G2 phase accumulation. Patients showing no response to radiotherapy had a high level of S-phase cells compared with partial (P < 0.001) and complete responders (P = 0.016). An inverse relationship was found when analysing the fraction of cells in G2 (P = 0.009 and 0.034 respectively). In general, healthy donors had similar cell cycle kinetics compared with the non-responders. In conclusion, the result indicates that radiation-induced cell cycle delay in lymphocytes is inversely correlated with tumour response to radiotherapy in head and neck cancer patients. However, the value of the present test for predicting individual tumour response is limited, because of assay variability and overlap between groups.

Cell cycle perturbations in three lung carcinoma cell lines (U-1285,U1906 and U-1810) with different intrinsic radiosensitivities (SF2 U-1285 = 0.25, SF2 U-1906 = 0.45, SF2 U-1810 = 0.88) were investigated following x-irradiation. Cell cycle flow calculations showed that the G1-->S-phase transit was accelerated in irradiated compared with untreated U-1285 cells, up to 24 hours postirradiation. In U-1810 cells and U-1906 cells the postirradiation G1-->S transit decreased compared with controls. All three cell lines showed no postirradiation induction of p53 and p21CIP1 proteins. Cyclin E was overexpressed and cyclin E-dependent kinase activity was substantially induced by irradiation in U-1285 cells compared with U-1906 and U1810 cells while p27KIP1 was detected at the highest intensity in U-1810 cells and lowest in U-1285 cells. We hypothesise that the accelerated postirradiation G1-->S transit in U-1285 cells is associated with induction of cyclin E-dependent kinase activity and may account for increased radiosensitivity in these cells.

Previous studies have indicated that transcription of germ-line (GL) CH genes is necessary to obtain immunoglobulin (Ig) class switching. We report here a correlation between proliferation, switching and GL transcripts. Smu-S gamma 1 switch recombination in lipopolysaccharide (LPS) + interleukin-4 (IL-4)-activated mouse B cells was assayed by a digestion-circularization polymerase chain reaction. Switching to gamma 1 is reduced upon inhibition of DNA synthesis with hydroxy-urea (HU) or aphidicholin (AC). Incubation of activated B cells with HU severely reduces steady-state levels of GL gamma 1 and epsilon RNA. By utilizing elutriation to synchronize B cell blasts in different phases of the cell cycle, it was found that GL gamma 1 transcripts are mainly expressed in G1 and S phases, but not in G0. Using the electrophoretic mobility shift assay, we characterized two major LPS-induced complexes, which bind to the GL gamma 1 promoter and are expressed at levels which correlate with the amount of LPS-induced DNA synthesis. Furthermore, the intensity of the complexes is reduced when cells are arrested with the DNA synthesis inhibitors HU or AC. Elutriation experiments revealed that the complexes are expressed in G1 and S, but not in G0. They bind to an Ets consensus element near the major initiation sites used in proliferating cells. The possible implications of these findings for Ig isotype switching are discussed.

Exposure of a myeloma cell line (RPMI 8226) to a 30-minute pulse of melphalan (1-phenylalanine-mustard) resulted in a cell cycle progression delay characteristic for DNA cross-linking agents. Reduction of outflow of cells from late S- and G2-phases was more pronounced as compared to that from G1-phase. The consequence is a progressive accumulation of cells in late S- and G2-phases. At restoration of outflow of cells from late S- and G2-phases, complete removal of DNA interstrand cross-links, as measured by DNA alkaline elution, was noted. At this time less than 50% of maximum DNA-protein cross-links were removed. Further we found no correlation between restored outflow of cells from the G2-phase and removal of DNA-protein cross-links during the follow-up time of 72 h. No DNA double strand breaks as measured by DNA neutral elution were formed during the observation period. The data suggest that removal of DNA interstrand cross-links seems prerequisite for the outflow of cells from G2 after melphalan treatment.

Human peripheral lymphocytes activated with concanavalin A and phorbol myristate ester exhibit an increase in glycolysis on a time-course similar to that for DNA synthesis. Elevated glycolysis is accompanied by increased specific activities of the glycolytic enzymes. Increased enzyme activities are accounted for by the appearance of specific isoenzyme forms (muscle forms) normally expressed in rapidly growing tumor cells or in growth-stimulated cells. In the present study we analyzed the expression of the glycolytic isoenzymes during cell cycle progression of activated human lymphocytes using two-parameter (DNA and protein) flow cytometry. Time-course studies and analysis of subpopulations prepared by elutriation centrifugation showed that the inducible isoenzymes are expressed at low levels or not at all in G0 cells. They are expressed first during the G0 to G1 transition or in early G1. However, expression increases throughout G1, reaching a maximum in S-phase. Thus, induction of glycolytic isoenzymes provides an excellent marker of T-cell activation and progression toward DNA synthesis.

Thymidine kinase (TK) activity was measured in relation to the cell cycle of in vivo growing ascites tumour cells. The cells were synchronized by means of centrifugal elutriation and the cell cycle composition of the cell fractions was determined by flow cytometry. TK activity was low in G1, increased during S phase and declined in G2. A half-life of TK activity of about 45 min was found throughout the cell cycle. Four isoenzymes at pI values of 4.1, 5.3, 6.9 and 8.3, denoted as isoenzymes 1-4, were identified using isoelectric focusing. Isoenzymes 3 and 4 were responsible for the profound cell cycle related changes in the TK activity. Corresponding isoenzymes were also found in the fetal mouse liver. In the adult mouse liver isoenzyme 2 was the dominating isoenzyme. The half-life of the isoenzymes was in the same range as for the total TK activity. We conclude that the low TK activity in G1 is due to degradation of the enzyme in G2 at a normal rate combined with an arrest in the synthesis of TK. We also conclude that isoenzyme 4 and the intermediate isoenzyme 3, which had earlier been suggested to be a mitochondrial form of TK, in fact represent cytoplasmatic forms of TK. According to cell cycle and pI studies, isoenzyme 2 belongs to the mitochondrial form. Studies with various phosphor donors and specific substrates, however, indicate that it also contains a cytoplasmic component.

The effect on cell growth and cell cycle kinetics of 0.8 mg cisplatin (CDDP)/kg body weight and 36 mg 5-fluorouracil (5-FU)/kg body weight given separately and in combination was studied on Bp8 mouse ascites sarcoma growing in vivo. Cell growth inhibition after combined treatment was delayed 12 hours but was persistent, while the cell growth inhibition was immediate after single drug treatment with a relative cell regrowth observed at the end of the observation period. The prolonged cell growth inhibition after combined treatment was probably due to cell death. Some cell kinetic interactions were found after combined treatment. An increased flow of cells from G1 was observed during the whole observation period. The depressed outflow of cells from S after single drug treatment was abolished during the first 24 hours following combined treatment. No prolongation was found on the CDDP-induced G2 delay. An increased relative number of cells in G2 following combined treatment was, however, found at 72 hours. This was due to an increased flow of cells from S to G2 seen after 48 hours. The molecular reasons and consequences are discussed.

The association between DNA precursor synthesis, cell cycle perturbations, and cell death caused by the anticancer drug hydroxyurea was investigated in mouse and human T-lymphoma cells. Hydroxyurea inhibits the enzyme ribonucleotide reductase, leading to decreased deoxyribo nucleoside triphosphate pools and an accumulation of cells in early S-phase of the cell cycle. We wished to clarify the mechanism of cell death caused by hydroxyurea in concentrations that can be obtained therapeutically. At a 60-microM concentration of the drug, giving 25% growth inhibition during 24 h, no increase in the number of dead cells was observed as determined by cell flow calculations and density gradient centrifugation. However, the removal of hydroxyurea led to 10-30% cell loss during the following 12-h period. In parallel, there was an increase in DNA precursor levels and a rapid progression of cells through S- and G2 phases of the cell cycle. The isolated dead cells showed no overrepresentation of any cell cycle phase. The results demonstrate that, although the toxic effects of low concentrations of hydroxyurea are minimal, the drug-induced unbalanced growth state can cause substantial cell death during a posttreatment period.

The cell flow and cell loss of an in vivo growing Ehrlich ascites tumour were calculated by sequential estimation of changes in the total number of cells in the cell cycle compartments. Normal growth was compared with the grossly disturbed cell flow evident after a 5 Gy X-irradiation. The doubling time of normal, exponentially growing cells was 24 hr. The generation time was 21 hr based on double-isotope labelling studies and the potential doubling time was 21 hr. Thus, the growth fraction was 1.0 and the cell loss rate about 0.5%/hr. Following irradiation, a transiently increased relative outflow rate from all cell cycle compartments was found at about 3 and 40 hr, and from S phase at 24 hr after irradiation. Minimum flow rates from all compartments were found up to 20 hr. Cell loss as calculated from the cell flow was compared with non-viable cells determined by Percoll density separation. Increase in cell loss as well as non-viable cells was observed at 24 hr after irradiation at the time of release of the irradiation-induced G2 blockage. Up to 50 hr, about 70% of the initial total number of cells were lost. The experiments show the applicability and limitations of cell flow and cell loss calculations by sequential analysis of the total number of cells in the various parts of the cell cycle.

Split dose irradiation with 2 X 2.5 Gy at intervals of 3 to 48 h was compared with the effect of a single irradiation with a 5 Gy dose in Bp8 mouse ascites sarcoma growing in vivo. The total number of cells and, using rapid-flow cytofluorometry, the proportions of cells in the various parts of the cell cycle were determined up to 10 days after irradiation. In addition, from sequential analyses of the total number of cells in the various compartments of the cell cycle, the flow of cells through the cell cycle was calculated. As judged by the growth in the total number of cells, a rapid repair of at least 20 per cent per h up to 3 h is followed by a slow repair of about one per cent per h during the following 45 h. The proportion of cells in the cell cycle following the conditioning dose did not differ enough to expect a significant influence on the mean radiation sensitivity of the cell population. At split time intervals of 12 to 48 h, after a transient early increase of the flow of cells through the cell cycle, the flow of cells was markedly reduced in all parts of the cell cycle with a maximum 24 to 48 h after the second dose of irradiation. This increase in the cell cycle time was not found following single dose irradiation and may influence the repair of potentially lethal cell damage.

DNA precursor synthesis can be blocked specifically by the drug hydroxyurea (HU) which has therefore been used for anticancer therapy. High concentrations of HU, however, affect other processes than DNA synthesis; nevertheless, most studies on the biological action of HU have been made with concentrations at least one order of magnitude higher than those needed for cell-growth inhibition. In this study we characterized the effects of low concentrations of HU (i.e. concentrations leading to 50% inhibition of cell growth in 72 h) on cell cycle kinetics and nucleotide pools in mouse S49 cells with various defined alterations in DNA precursor synthesis. The effect of 50 microM HU on deoxyribonucleoside triphosphate pools was a 2-3-fold decrease in the dATP and dGTP pools, with no change in the dCTP pool and a certain increase in the dTTP pool. Addition of deoxycytidine or thymidine led to a partial reversal of the growth inhibition and cell-cycle perturbation caused by HU, and was accompanied by an increased level of the deoxyribonucleoside triphosphates. Addition of purine deoxyribonucleoside gave no protection, indicating that salvage of these nucleosides could not supply precursors for DNA synthesis in T-lymphoma cells. We observed a higher sensitivity to HU of cells lacking purine nucleoside phosphorylase or with a ribonucleotide reductase with altered allosteric regulation. Cells lacking thymidine kinase or deoxycytidine kinase were just as sensitive as wild-type cells.

The size of the dCTP pool has been implicated as a possible regulator of DNA synthesis. In this investigation we correlate large intracellular variations in deoxyribonucleoside triphosphate levels to the growth rates and cell-cycle kinetics of mouse S49 T-lymphoma cells. Wild-type and a mutant line AzidoC-100-5, lacking dCMP-deaminase activity resulting in a 10-fold expanded dCTP pool were studied and compared using flow cytometry, centrifugal elutriation and nucleoside triphosphate determinations. An increase in the dCTP pool was closely correlated to the passage of cells from G1 to S phase in both cell types. Addition of thymidine to wild-type and mutant cells resulted in an accumulation of cells in early S phase, concomitant with a decreased dCTP level. Mutant cells excreted large amounts of deoxycytidine into the medium which partially protected the cells from thymidine inhibition. The doubling times for the mutant and wild-type cells were very similar but the mutant had a somewhat prolonged S phase and shortened G1 phase compared with the wild-type cells. Large changes in the DNA precursor levels were produced by addition of thymidine to mutant cultures. This gave no change in the growth rate but a somewhat shortened S phase and prolonged G1. The biochemical background for these effects is discussed.

Bp8 ascites sarcoma cells growing in vivo were whole body irradiated with doses of 1.75 to 8 Gy. The inflow and outflow rates of cells in the various compartments of the cell cycle were estimated on the basis of sequential analysis of the total number of cells and from the proportion of cells in G1, S-phase, G2 and M. The flow through the compartments was calculated from these data and from the sizes of the cell pools. The durations of the mitotic delay, the G1-depletion and the early G2 blockage were linearly related to the dose. After release of the mitotic division delay the accumulation rate of cells in M and G1 decreased linearly with the dose; for G2, plateau values for the accumulation rate were found after 2.5 Gy. The outflow rates from G2 and M after release of the G2 blockage showed a shoulder type of dose-response with a D0 of 2.5 Gy and a n value of 1.5. In addition to an increase in the duration of G2, generally responsible for the increase in the total cell cycle time at about 24 hours after irradiation and an increase in the duration of the S-phase up to 12 hours after irradiation, an early increase in the duration of G1 and M was observed.

The cell volume of Ehrlich ascites tumour cells was studied following a radiation dose of 5.0 Gy. The cell volume increased 12 to 30 hours after irradiation by about 20 per cent, was normal at about 50 hours, and increased again at 72 hours. In order to explain these changes the composition of the cells in cell cycle was studied. In addition, the cell volume of irradiated cells from the various parts of the cell cycle, separated by centrifugal elutriation, was measured. The changes in the mean cell volume of unseparated cells could be explained by variations in the cell cycle composition of the cell population. Irradiated cells from the various parts of the cell cycle did not deviate from the volume of non-irradiated cells. The cell volume doubled during the cell cycle. This increase was, however, not linear.

The mechanism of toxicity of foscarnet was studied by monitoring its effects on the cell cycle of exponentially growing, semisynchronous human embryo cells in culture. The effects of foscarnet on the cell cycle were dependent on the concentration of drug used. At 1 mM, cell division was reduced by 50%, whereas the cell flow was mainly reduced in the G2 phase of the cell cycle, leading to an increase in the proportion of G2+M cells. The minor reduction of thymidine incorporation in S phase cells provided additional evidence that 1 mM foscarnet did not specifically inhibit DNA synthesis. Cell division was greatly reduced at 2.5 mM foscarnet, and the G2 phase was markedly affected, whereas S cell flow was less reduced. S cell flow was 10% per h and thymidine incorporation was 25% that of control cells, while a block in the G2+M phase was evident. On the other hand, at a concentration of 5 mM foscarnet, the cell flow was greatly reduced in the G1 and S phases, with less reduction of G2 cell flow and cells accumulated in the S phase. The effects of foscarnet on the cell cycle were more pronounced with increasing times up to 72 h, which could not be explained by the slow penetration of foscarnet which required only 4 to 8 h to achieve constant levels. At 2.5 and 5 mM foscarnet, there was the additional effect of the cell membranes becoming more leaky as a result of foscarnet toxicity which might contribute to the toxic effects of the drug at high concentrations. When foscarnet was removed from the medium, the effects on the cell cycle were rapidly reversed, in the time needed for foscarnet to diffuse out from the cells, which indicates the reversible nature of the toxic effects of foscarnet.

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.

Consistent with its specialized role in DNA synthesis, the activity of ribonucleotide reductase is cell cycle-dependent, reaching its maximum during S-phase. This paper demonstrates, however, the levels of the two protein subunits, M1 and M2, of this enzyme vary independently of one another. The level of protein M1 was determined by use of a two-site monoclonal antibody-enzyme immunoassay and found to be constant throughout the cell cycle in bovine kidney MDBK cells. Pulse-chase experiments showed that the half-life of protein M1 was 15 h. This contrasts with our previous results demonstrating an S-phase-correlated increase in the concentration of protein M2 and a half-life of this subunit of 3 h. Therefore, ribonucleotide reductase is controlled during the cell cycle by the level of protein M2.

Abstract. Human DNA-dependent protein kinase (DNA-PK) is a nuclear-localized serine/threonine protein kinase. The holoenzyme consists of a catalytic subunit with a molecular mass of 465 kDa and a DNA-binding heterodimer Ku86/70. The kinase has been implicated in a variety of nuclear processes including V(D)J recombination, double-strand break repair, and transcription. Cells with defective DNA-PK activity show increased radiosensitivity and lack of V(D)J recombination. To study DNA-PK activity during the cell cycle, HeLa cells were separated by elutriation centrifugation into different cell cycle compartments based on cellular size. DNA-PK activity was found to vary during the cell cycle. The kinase activity was lowest during G1 phase and increased dramatically as the cells entered S phase and remained high during the G2-phase. The subcellular distribution of DNA-PKcs is relocalized from the cytoplasm during M and G1 phases to the nucleus during G1–S phase transition and S phase. Expression of both the catalytic subunit and the Ku86/70 heterodimer was found to be constant throughout the cell cycle. This study demonstrates that DNA-PK activity as well as its subcellular localization fluctuates during the cell cycle. In addition, the distribution of DNA-PK during M phase corresponds with low DNA-PK activity. [ABSTRACT FROM AUTHOR]