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   1   Polysaccharopeptide enhances the anticancer activity of doxorubicin and etoposide on human breast cancer cells ZR  - 75 -  30    Jennifer Man-Fan Wan, Wai-Hung Sit and Jimmy Chun-Yu Louie School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P.R. China  Abstract In search of natural bioactive microbial compounds with adjuvant properties, we have previously showed that the polysaccharopeptide (PSP), isolated from Chinese medicinal mushroom Coriolus versicolor  , was able to enhance the cytotoxicity of certain S-phase targeted-drugs on human leukemic HL-60 cells via some cell-cycle and apoptotic-dependent pathways. The present study aimed to investigate whether the synergism of mechanisms of PSP with certain chemotherapeutic drugs also applies to human breast cancer. PSP treatment enhanced the cytotoxicity of doxorubicin (Doxo), etoposide (VP-16) but not cytarabine (Ara-C). Bivariate bromodeoxyuridine (BrdUrd)/DNA flow cytometry analysis estimated a longer DNA synthesis time (Ts) for the PSP treated cancerous cells suggesting that PSP enhanced the apoptotic effect of Doxo and VP-16 via creating an S-phase trap in the human breast cancer cell line ZR-75-30. The participation of PSP in the apoptotic machinery of the chemotherapeutic agents was further supported by a reduced ratio of protein expression of Bcl-xL/Bax of the cancer cells. This study provides further insight into the synergistic mechanisms of PSP and supports the hypothesis that the anti-cancer potentials of PSP is not limited to leukemia but may also be used as an adjuvant therapy for breast cancers. Key words:  polysaccharopeptide, bromodeoxyuridine, doxorubicin, etoposide, cytarabine, human breast cancer, Chinese medicinal mushroom Int J Oncol.  2008; 32(3):689-99. INTRODUCTION Breast cancer is one of the most frequent cancers in the world (1), and it is the commonest cancer amongst women (1,2). The mortality of breast cancer is low (1), however, because of its high incidence and the increasing global trend (1,3,4), it results in medical costs worldwide estimated to be more than US$7 billion (3). The absence of estrogen receptor (ER) is found to be associated with higher recurrence rate (5), and patients with an ER-negative status have a significantly shorter median survival time (6). Hormonal therapy, one of the treatment used to tackle breast cancer, is reported to be completely ineffective in ER-negative breast cancers (7), therefore rendered the high importance of chemotherapy in ER-negative breast cancer patients. However, chemotherapy comes with side effects such as alopecia (8) and leucopenia (9) because it does not differentiate between cancerous cells and normal cells (9-15). Many efforts have been put into the search for anti-tumor agents that can distinguish cancerous cells from normal cells, as well as adjuvant therapies that can enhance the cytotoxicity of the anti-tumor drugs to cancerous cells, which give potential of lowering chemotherapeutic drugs to reduce the side effects. Combined chemotherapy has also been tried in the hope of reducing the drug-induced side effects as well as increasing the anti-tumor specificity.  When searching for natural anticancer compounds for combination therapy, it is essential to identify novel compounds that are capable of potentiating the chemotherapeutic value of the anticancer drugs but at the same time exert no cytotoxic effect to normal cells. In this respect, the polysaccharopeptide (PSP), isolated from the mycelium of the medicinal Chinese mushroom of the species Coriolus versicolor  , known as Yun Zhi, offers great potential in cancer combined treatment therapy because of its ability to distinguish cancerous cells from normal cells (16-20). PSP is currently in phase II clinical trials in China, with great success including high survival rates, improved immunological activities, appetite and comfort for patients status (19). The anticancer mechanisms of PSP include induction of apoptosis in cancer cells (21-24) and enhancement of host immune response (24-28).  The active component of Yunzhi, polysaccharopeptide (PSP), consists of 6 types of monosaccharides, namely mannose, glucose, xylose, galactose, arabinose and rhamnose, connected  with a small polypeptide (17). Fig. 1 gives an illustration of the partial structure of the polysaccharide moiety of PSP. The polypeptide moieties of PSP are rich in aspartic and glutamic acids (Table I) (17-19). In a recent study, we have demonstrated that PSP was able to potentiate the chemotherapeutic activities of certain cell cycle-specific anticancer drugs. When combined with S-phase-specific chemotherapeutic drugs, we observed that PSP  was effective in potentiating the cytostatic and apoptotic effects of both doxorubicin (Doxo) and etoposide (VP) on the human leukemic HL-60 cells (24). In the present study, the ZR-75-30 cancer cell line, an estrogen receptor (ER) negative human breast cancer was chosen in order to investigate whether PSP can potentiate the cytostatic and apoptotic effects of doxorubicin, etoposide and cytarabine on human breast cancer.  The 5'-Bromo-2'-deoxyuridine (BrdUrd) labeling of DNA technique with flow cytometry (29-33) was applied to investigate the cell cycle-specific actions of PSP and its interaction with Doxo, VP-16, and Ara-C. The present study showed that the combined treatment of S-phase targeting drugs with PSP also resulted in higher cytotoxicity than drugs given alone in the ZR-75-30 cancer cells. Flow cytometry analysis indicates that PSP enhanced the cytotoxicity of the tested drugs by creating an S-phase trap in ZR-75-30 cells. This study provides further insight into the synergistic mechanisms of PSP and supports the hypothesis that the anticancer potentials of PSP is not limited to leukemia but may also be used as an adjuvant therapy for breast cancers. MATERIALS AND METHODS Reagents.  Cytarabine (Ara-C), doxorubicin (Doxo), dimethyl sulfoxide (DMSO) and protease inhibitor cocktail were purchased from Sigma. Etoposide (VP-16) was purchased from Calbiochem. Polysaccharopeptide (PSP) was obtained from the Winsor Health Products Ltd. in form of capsules (340 mg/capsule). The crude powder of PSP was dissolved in distilled  water, and the water soluble fraction was then freeze-dried. The freeze-dried powder was then dissolved in distilled water to prepare a solution at concentration of 10 mg/ml. Stock solutions of Ara-C and Doxo were prepared in distilled water, and VP-16  was dissolved in DMSO. Annexin V binding assay easy to use detection kit (Apoptosis Detection Kit) was obtained from  Trevigen Inc. Monoclonal anti-human Bcl-2, anti-human Bax, anti-human Bcl-xL and anti-human Bid antibody were supplied from Santa-Cruz Biotechnology. Cell culture  . ZR-75-30 cell line used in this study is an estrogen receptor (ER)-negative human breast cancer (34) derived from the ascite fluid of a 47-year-old post-menopausal Black women  with infiltrating duct carcinoma (35) and it was a generous donation of Dr Mabel Young from the Department of Physiology, the University of Hong Kong. ZR-75-30 was routinely culture in RPMI medium supplemented with 10% fetal bovine serum. The densities of the cells were monitored to ensure they did not exceed 2x107 cells per 80 cm2 culture flask.   2   Cells were cultured at 37°C in a humidified atmosphere with 5% CO 2 . The cultures were supplied with fresh complete medium and the cell density was adjusted to 1x10 6  cells per flask every three days to maintain asynchronous and exponential growth. In all experiments, they were fed with fresh complete medium a day before the experiment. Cell proliferation analysis  . The time (24, 48, 72 h) and dose dependent (0-400 μ g/ml) effect of PSP on the proliferation of ZR-75-30 human breast cancer cells were studied by culturing the cells with or without (RPMI only) PSP as described above. Preparation of PSP has been described elsewhere by us (16,24).  After trypsinization, 100 μ l cancer cell aliquots and 100 μ l of  Trypan-blue solution was added to a 1.5 ml microcentrifuge tube  which was then mixed thoroughly and incubated at room temperature for 5 min. Total number of cells in four 1 mm 2  corners of hemacytometer was counted, and the average number of cells per unit volume of medium was calculated. Study 1: the influence of cell cycle kinetics of PSP on ZR-75-30 human breast cancer cells measured by bivariate BrdUrd/DNA flow cytometry  . Previous report with human leukemic cells (16,24) indicated that one of the anticancer properties of PSP involves S-phase cell cycle arrest. This study sought to investigate the influence of cell cycle kinetics of PSP on the ZR-75-30 human breast cancer cells by using the bromodeoxyuridine labeling and bivariate BrdUrd/DNA technique analysis by flow cytometry.  To investigate the effect of PSP on tumor growth and cell cycle distribution (G0/G1, S and G2/M), ZR-75-30 cell suspensions at 2x106 cells were exposed with or without PSP at concentrations ranging between 5 and 400 μ g/ml. The cells were incubated for 24, 48 or 72 h, harvested and examined by various analyses. The fixed samples were stored at - 20˚C for further analysis by DNA/ PI flow cytometry. To study the effect of PSP on the cell cycle kinetics [labeling index (LI), relative movement (RM), DNA synthesis time (Ts) and G 0 /G 1  cells returned or labelled divided cells (Lud)], ZR-75-30 cells with 48-h pre-treatment of PSP (50 μ g/ ml) or without (control) were pulsed with 10 μ M of BrdUrd for 20 min. After pulse-labelling with BrdUrd, cells were washed  with warm cell culture medium, divided and subjected to culture incubation for 1 or 6 h. At the end of incubation, cells were harvested and fixed with ice-cold 75% ethanol. The fixed samples  were stored at - 20˚C for further analysis by flow cytometry.  The bivariate BrdUrd/DNA technique analysis by flow cytometry is based on simultaneous measurement of the total incorporation or the rate of incorporation of the thymidine analog, BrdUrd, into newly synthesized DNA (30-32). The uptake of BrdUrd in each cell can be detected by immunocytochemical staining techniques that involve binding the BrdUrd to fluorescein isothiocyanate (FITC) labeled anti-BrdUrd monoclonal antibodies (green fluorescence). The quantity of BrdUrd (green fluorescence) and DNA (red fluorescence labels by propidium iodide) can be simultaneously measured in the same cells. This analysis estimates the relative number of cells actually involved in cell division (labelling index) and may also provide estimates of the rate of DNA synthesis. By repeating the bivariate BrdUrd/DNA analysis of labelled cells at several time-points, it is possible to determine the rate of progression of BrdUrd-labelled cells as they accumulate more DNA. The movement of the BrdUrd-labeled cells (cycling cells) between the G0/G1 and G2/M cell cycle phases can be used to calculate the ‘relative movement’ (RM). By using two different relative movements (RM(t)) extracted from the different BrdUrd pulse-labelling time, the DNA synthesis time (Ts) of the cancer cells can be calculated according to the methods previously described (29-33): RM(t) = (F lud(t)  - F G0/G1  )/(FG 2/M  - F G0/G1  )  Ts = 0.55(t)/(RM(t) - RM(0))  where F lud(t) , F G0/G1  and F G2/M  are the mean PI fluorescence (DNA content) of the BrdUrd labelled undivided cells of the G 0 /G 1  and G 2 /M population at time (t) (hour) after BrdUrd pulsing. RM(0) and RM(t) are the relative movement values at time 0 and t (hour) after BrdUrd pulsing respectively. RM(0) is estimated to be 0.55. Immunocytochemical staining for BrdUrd/DNA flow cytometry analysis.  Staining of cancer cells for bivariate analysis of BrdUrd was performed as previouly described by us (29,33,36) and by other investigators (30) with some modifications. Cells were harvested into 15 ml centrifuge tubes. All samples were washed twice with 10 ml PBS and centrifuged at 400 g for 5 min. Supernatant was removed and cell pellets were re-suspended in 100 μ l ice-cooled PBS. Cells were fixed by adding ice-cold EtOH (70% w/v in Milli-Q water) dropwisely into the tubes while the cells were being  vortexed. The fixed samples were stored at - 20˚C for further Figure 1 . The partial structure of polysaccharide moiety of PSP (17).   Amino acid Content (%) Amino acid Content (%)  Aspartic acid 4.0 Methionine 0.4 Glutamic acid 5.8 Isoleucine 2.2  Threonine 2.3 Leucine 2.4 Serine 3.2 Tyrosine 1.5 Proline 1.0 Phenylalanine 1.5 Glycine 2.6 Tryptophan 1.7  Alanine 2.6 Lysine 2.3 Cystine 0.9 Histidine 0.7  Valine 1.8 Arginine 1.8  Table I.  Amino acids composition in polypeptide moiety of PSP (17).    3   analysis. The fixed samples were washed twice each with 10 ml PBS and the DNA of cells were partially denatured by incubation  with 2 M HCl (0.5 ml) for 30 min. After denaturation, cells were  washed three times with 10 ml PBS containing 0.05% Tween-20 (PBS-T). The cells were incubated with 100 μ l of anti-BrdUrd antibody (1:100 dilution) and incubated at room temperature for 1 h. After incubation, cells were washed twice with PBS-T and subsequently incubated with 100 μ l of FITC-conjugated anti-mouse IgG antibody (1:40 dilution) to label the primary antibodies in dark at room temperature for 1 h. Following the incubation, cells were washed twice with PBS and the DNA of cells was stained with propidium iodide (PI) staining solution (50 μ g/ml PI, 10 μ g/ml RNase, 0.01 M Tris-base and 10 mM NaCl in milli-Q water) for 30 min at room temperature. The stained cells were analyzed by Coulter's Epics Elite ESP flow cytometer at 525 nm and 620 nm band pass filters. Study 2: The combined anticancer effect of PSP with doxorubicin (Doxo), etoposide (VP-16) and cytarabine (Ara-C). Based on the cell cycle kinetic study of study 1, the testing hypothesis of this study was that by creating an S-phase trap, PSP can enhance the cell killing effect of chemotherapeutic agents namely doxorubicin, etoposide and cytarabine with S-phase interference activity on the ZR-75-30 cells. The ZR-75-30 cells were treated with PSP (50 μ g/ml) or  without (non PSP treated) for 48 h before adding the individual tested drugs. Both PSP pre-treated and non-PSP treated cells  were exposed to 5 μ M of Ara-C, VP-16 or Doxo for further 18 h.  The total treatment time of PSP was 66 h. Cells were harvested for annexin V/PI flow cytometry analysis for cell death and  Western blot analysis of the Bcl-2 family genes.  Quantification of cell death by annexin V/PI flow cytometry  .  The theoretical background of this detection method has been previously described (37). Annexin V binding assay was performed by using an apoptosis detection kit. Cells (2x10 5  ) after incubation with or without PSP and drugs were harvested and centrifuged at 400 x g for 5 min to remove culture medium. Cell pellets were washed with 3 ml phosphate buffer saline (PBS) and re-suspended in 500 μ l binding buffer. After centrifugation and removal of binding buffer, 100 μ l of annexin V incubation reagent (10 μ l 10X binding buffer, 10 μ l propidium iodide (PI), 1 μ l annexin V conjugate and 79 μ l milli-Q water) was added to each sample. The samples were incubated for 15 min in the dark at room temperature. The cell suspension was then diluted with 400 μ l binding buffer and was analyzed by flow cytometer. Western blot analysis of the expression of Bcl-2 family proteins. Cells were harvested and washed twice with ice-cold PBS (45 ml) followed by centrifugation at 400 x g for 5 min. Cell pellet was re-suspended in lysis buffer (5x106 cells/100 μ l lysis buffer) with HEPES (25 mM; pH 7.5), NaCl (150 mM), EDTANa2 (1 mM), DTT (1 mM), Triton X-100 (1%) and protease inhibitor cocktail.  The suspension was then frozen and thawed three times by cold methanol at - 80˚C. The iced suspension was sonicated to melting point. Cell suspension was then placed in ice for further 30 min and then centrifuged. After centrifugation at 14,000 x g for 30 min at 4˚C, the suspension was collected and stored at - 80˚C. Protein quantity was determined by Bradford assay. Protein extracts were mixed with equal volume of 2X sample buffer (0.125 M Tris-HCl, 4% SDS, 20% v/v glycerol, 0.2 M DTT, 0.02% bromophenol blue, pH 6.8) and the mixture was boiled in  water for 3 min. Equal amounts of total protein (20 μ g) were subjected to 12.5% SDS-PAGE followed by Western blotting onto a PVDF membrane. Membranes were incubated with anti-human Bcl-2, anti-human Bax, anti-human Bid and anti-human Bcl-xL antibodies, and detected with the matching species-specific secondary HRP-conjugated antibodies. Proteins were detected using the ECL system (GE Healthcare) and the band intensity  was measured by Quantity One software (Bio-Rad). Statistical analysis. All data are presented as mean ± standard error of the mean (SEM). Statistical significance was calculated using two-tail Student's t-test for two groups and One-way ANOVA analysis for multi-group comparison. p<0.05 was considered as statistically significant. RESULTS The anti-proliferation effect of PSP on ZR-75-30 cells.  Treatment with PSP delayed the proliferation of ZR-75-30 cells in a dose-dependent (Fig. 2a) and time-dependent (Fig. 2b) manner. At 50 μ g/ml of PSP, proliferation was decreased by 36.8% after 48-h treatment. Measured by annexin V/PI flow cytometry, at 72-h treatment, PSP induced significant cell death (p<0.001) by 23.9% (Fig. 3).  Effect of PSP on the human breast cancer ZR-75-30 cell cycle distribution.  The cell cycle distribution of ZR-75-30 cells, measured by DNA/PI flow cytometry, shows that PSP was capable of inhibiting the cell proliferation via alteration of cell cycle. At 48 h, PSP induced cell arrest in the S-phase by 34% (p<0.01) with a corresponding decrease of cell proportions in G0/G1 and G2/M phases (Fig. 4) compared to control.  Effect of PSP on labelling index (LI), relative movement (RM), and DNA synthesis time (Ts) of human breast cancer ZR-75-30 cells  . Fig. 5 presents the contour plot of the bivariate BrdUrd/DNA flow cytometry analysis from the 1- and 6-h BrdUrd pulse labelling of the PSP treated and control (non-PSP treated) cells. The S-phase BrdUrd labelled cell population of the 1-h BrdUrd contour plot is used to estimate the labelling index (LI) of the cancer cells as described (29-33). The 6-h contour plots show that the BrdUrd labelled cells have moved through the S-phase in the cell cycle and some had not yet divided (Lud) whereas others had done so. The newly divided daughter cells population detected by anti-BrdUrd-monoclonal antibodies of the G0/G1 phase was used to calculate the labelled divided cells (Ld). The relative movement calculated from the 6-h BrdUrd/DNA flow cytometry (RM 6) (Fig. 5) was used to calculate the DNA synthesis time (Ts) of the cancer cells.  Table II summarized the effect of PSP on LI, RM, Ld and Ts of the cancer cells with and without PSP treatment. The data show that PSP arrested cells in S-phase and resulted in significant higher calculated labelling index. It is noteworthy that PSP treatment significantly (p<0.01) retarded the relative movement (1 and 6 h) of the cancer cells. PSP arrested cells in S-phase resulted in a hyper-prolongation from 12.51 to 18.31 h for the DNA synthesis of the cancer cells. The DNA synthesis time of ZR-75-30 cells was extended by 46.4% (p<0.01). Calculation of the labelled divided cells also dropped from 8.58 to 3.67% suggesting some interference might have occurred at the G 2 /M phase. Figure 2.  The time and dose effect of PSP on the proliferation of human breast cancer ZR-75-30 cell proliferation. (a) dose-dependent effect; (b) time-dependent effect.    4   The effect of anti-tumour drugs alone and PSP pre-treatment with anti-tumor drug on cell death and cell viability in ZR-75-30.  To inuvestigate the interaction of PSP with the chemotherapeutic drugs, cell death  was performed with annexin V/PI flow cytometry. Fig. 6 indicates that comparing to the cancer cells administered only the corresponding individual chemotherapeutic agent treatment, the apoptotic effect of Ara-C, Doxo and VP-16 on the ZR-75-30 cells was further enhanced in cells with PSP pre-treatment by 18.8%, 55.4% (p<0.001) and 161% (p<0.001), respectively. The  viability of these cells dropped by 8.8, 33.2 (p<0.001) and 48.8% (p<0.001) with Ara-C, Doxo, and VP-16, respectively.  Effect of PSP with and without Ara-C, Doxo and VP-16 on apoptotic  protein expression in human breast cancer ZR-75-30 cells  . Western blot analysis of protein level presented in Fig. 7 shows that among the apoptotic genes measured, Bax gene expression was the strongest in the ZR-75-30 cells. Fig. 8 summarizes the relative expression of the Western plotted proteins and shows that comparing to the control, PSP treatment alone increased the pro-apoptotic Bax expression by 107% while that of Bid was reduced by 45%. Expression of the anti-apoptotic protein Bcl-xL was reduced by 51%. When comparing the chemotherapeutic agents, they produced similar effects, i.e. increasing Bax expression and decreasing Bcl-xL and Bid expression. Doxo treatment increased Bax expression by 39% and reduced Bcl-xL and Bid expression by 39 and 62%, respectively. Ara-C treatment increased expression of Bax by 209% and decreased Bcl-xL by 45%. Expression of Bid was decreased by 70% as well. VP-16 treatment increased Bax expression by 220%, while Bcl-xL and Bid expression were decreased by 52 and 90%, respectively. Compared with the corresponding non-PSP treated groups, combination treatment of PSP with Doxo increased the expression of Bax by 92.1% and slightly decreased the expression of Bcl-xL by 24.6%. The change of bid expression in Doxo with PSP is insignificant. In the combination treatment of PSP with Figure 3.  The effect of PSP on cell death of ZR-75-30 cells. The cells without (control) and with PSP (50 μ g/ml) treatment for 48 and 72 h were subjected to annexin V/PI flow cytometry analysis. The dot plots representing data on viable cells (R3) and dead cells (R1, R2, R4) at 48 and 72 h, respectively. Values are mean ± SEM (n=4). ***p<0.001 compared with control.  Figure 4 . The effect of PSP on the cell cycle distribution. Cancer cells without (control) or with 50 μ g/ml PSP incubation for 48 h  were fixed with PI for staining of DNA. The cell population (%) distribution among the G0/G1, S and G2/M phase was analyzed by DNA/PI flow cytometry. Values are mean ± SEM (n=4). **p<0.01 compared with control.    5    VP-16, the change in Bax expression is insignificant while Bid expression was doubled. Bcl-xL expression was decreased by 35.4%. Combined treatment of PSP with Ara-C, to our surprise, reduced the expression of Bax by 9.4%, while Bcl-xL expression  was decreased by 21.8%. Change in Bid expression is insignificant. Fig. 8d shows that all treatment groups decreased the Bcl-xL/Bax ratio. PSP alone decreased the ratio by 76.45% while Ara-C, Doxo and VP-16 given alone decreased the ratio by 82.4, 56.0 and 84.9%, respectively. Compared with the corresponding non-PSP treated groups, PSP further decreased the Bcl-xL/Bax ratio induced by Doxo by 60.6%, Ara-C by 13.0% and VP-16 by 37.5%. DISCUSSION Breast cancer is one of the most frequent cancers in the world (1), and it is the commonest cancer amongst women (1,2). Because of the advancement in breast cancer therapy, the mortality is low compared to other types of cancers (1). Therapies used for breast cancer include hormonal therapy (7,38), chemotherapy (39), a combination of both (40), and surgical removal of the malignant tissue. Surgical removal results in psychological issues such as low self-esteem (41), which is highly undesirable. Hormonal therapy is reported to be ineffective in ER -negative breast cancers (7), therefore chemotherapy in ER-negative breast cancers is very important. Many chemotherapeutic agents, however, are also toxic to normal cells (10-15), leading to Figure 5.  The effect of PSP on the labelling index and labelled divided cells: measured by bivariate BrdUrd/DNA flow cytometry. Contour plots of bivariate BrdUrd/DNA flow cytometry distribution of the breast cancer cells without or with PSP (50 μ g/ml) treat-ment for 48 h were generated from the 1 and 6 h BrdUrd pulse labels, respectively. The S-phase cells situated in between 2N (G0/G1) and 4N (G2/M) labelled positively with BrdUrd, and this population of cells were used to estimate the labelling index (LI, %).  The 6-h BrdUrd/DNA contour plot shows that some S-phase cells incorporated BrdUrd and had progressed through the G2/M phase; further divided and appeared in G0/G1 as daughter cells or labelled divided cells (Ld). Some cells remained undivided and expressed as labelled undivided cells (Lud). Values are mean ± SEM (n=4). **p<0.01 compared with control.   Time (h) LI(%) Ld(%) RM Ts Control 1 31.9±0.4 0.66±0.01  PSP 1 42.8±1.3 a  0.59±0.01 a   Control 6 8.58±0.52 0.82±0.01 12.51±0.56  PSP 6 3.67±0.24 a  0.73±0.01 a  18.31±0.63 a    All data are expressed as mean ± SEM (n=4). a p<0.01 vs control; PSP (50 μ g/ml). LI, labelling index, extracted from the 1-h bivariate BrdUrd/DNA flow cytometric contour plot; Ld, labelled divided, extracted from the 6-h bivariate BrdUrd/DNA flow cytometric contour plot; RM, relative movement; extracted from both 1- and 6-h bivariate BrdUrd/DNA flow cytometric contour plot; Ts, DNA synthesis time; Ts = 0.55(t)/(RM(t) - RM(0)), where F lud(t) , FG 0 /G 1 and FG 2 /M are the mean PI fluorescence (DNA content) of the BrdUrd labelled undivided cells of the G 0 /G 1 and G 2 /M population at time (t) (hour) after BrdUrd pulsing; RM(0) and RM(t) are the relative movement values at time 0 and t (hour) after BrdUrd pulsing, respectively. RM(0) is estimated to be 0.55.  Table II.  The effect of PSP on labeling index, labeled divided cells, relative movement and DNA synthesis time of the ZR-75-30 cells. 
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