Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. II. Percentage labelled mitoses studies

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (Tc) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G1, S and G2 phases of the crypt cell cycle are altered by sympathectomy, G1 accounting for most of the increase in Tc. In addition, the percentage of [3H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

EXISTENCE OF TWO CHALONE-LIKE SUBSTANCES IN INTESTINAL EXTRACT FROM THE ADULT NEWT, INHIBITING EMBRYONIC INTESTINAL CELL PROLIFERATION

The inhibiting effect of tissue extract from fully differentiated intestinal mucosa of adult animals on proliferation kinetics of exponentially growing embryonic epithelial gut cell populations was studied in the newt Pleurodeles waltlii. Crude extract was fractionated by G-200 Sephadex chromatography and the effect of fractions on cell proliferation was studied using both mitotic index and ³H-thymidine incorporation methods. The inhibitions we obtained were then displayed by means of cytophotometric study of age distribution of intestinal gut cells around the cell cycle, measuring the Feulgen-DNA content. The results revealed the presence of two chalone-like substances in the intestine of adults. One (factor 1) is characterized by a molecular weight of between 120,000 and 150,000 and inhibits the cell cycle at the end of the G₁ phase, the other (factor 2) is characterized by a molecular weight lower than 2000 and inhibits the cell cycle in the course of the G₂ phase. The cells delayed in the G₂ phase escape from inhibition but the cells delayed in the G₁ phase do not, although availability time of both factor 1 and factor 2 is about 12 hr. It is thus thought that cells prevented from dividing in G₁ phase are indefinitely delayed in this phase and possibly differentiate.     

Bovine pancreatic trypsin inhibitor is a new antifungal peptide that inhibits cellular magnesium uptake

Antimicrobial peptides (AMPs) are promising agents for control of bacterial and fungal infections. Traditionally, AMPs were thought to act through membrane disruption but recent experiments have revealed a diversity of mechanisms. Here we describe a novel antifungal activity for bovine pancreatic trypsin inhibitor (BPTI). BPTI has several features in common with a subset of antimicrobial proteins in that it is small, cationic and stabilized by disulphide bonds. BPTI inhibits growth of Saccharomyces cerevisiae and the human pathogen Candida albicans. Screening of the yeast heterozygous essential deletion collection identified the magnesium transporter Alr1p as a potential BPTI target. BPTI treatment of wild type cells resulted in a lowering of cellular Mg²⁺ levels. Populations treated with BPTI had fewer cells in S‐phase of the cell cycle and a corresponding increase of cells in G₀/G₁ and G₂ phases. The same patterns of cell cycle arrest obtained with BPTI were also obtained with the magnesium channel inhibitor hexamine(III)cobalt chloride. Analysis of the growth inhibition of C. albicans revealed that BPTI is inhibiting growth via the same mechanism in the two yeast species. Inhibition of magnesium uptake by BPTI represents a novel mechanism of action for AMPs.     

THE CELL CYCLE TIME IN THE RAT JEJUNAL MUCOSA

The regional variation of the duration of cell cycle parameters was studied by constructing fraction of labelled mitoses curves at several levels in the jejunal crypt column of male Wistar rats. Prolonged T_c and T_s values were apparent only in the bottom eight cell positions, and these differences were shown to be significant compared with the remaining cell positions by analysing the data by the method of Gilbert (1972). Above cell position 8 the proliferating crypt cells showed effectively the same phase durations. For the whole crypt column T_c was 11.32 ± 0.14 (SE) and T_s 6.49 ± 0.10. Although variation in phase durations was confined to the basal portion of the crypt, the results essentially confirm the findings of Cairnie, Lamerton & Steel (1965a), and may be interpreted in terms of the slow cut-off model. The demonstration of prolonged T_c values in basal cell positions confirms the presence of a longer cycling subpopulation of cells at the bottom of the crypt.     

Effects of Interferon γ on growth,apoptosis, and MHC class II expression of immature rat intestinal crypt (IEC-6) cells

Intestinal epithelial cells and the mucosal immune cells in close proximity are thought to interact very closely. One well-established mechanism of this intercellular cross-talk is via the production of cytokines such as interferon gamma (IFNγ). The aim of this study was to analyze the effects of IFNγ on intestinal crypt epithelial cells. IEC-6 cells were cultured in the presence or absence of IFNγ to measure its effects on proliferation, cell cycle, apoptosis, and major histocompatibility complex (MHC) class II antigen expression. Even at very low doses (0.01 U/ml), IFNγ significantly inhibited IEC-6 cell proliferation, as demonstrated by reduced ³H-thymidine uptake, stable cell count, and complete arrest in the quiescent G₀/G₁ phase of the cell cycle. Incubation with supraphysiological doses of IFNγ (100–1,000 U/ml) did not induce apoptosis, as assessed by morphology and the TUNEL assay. IFNγ significantly induced de novo IEC-6 class II antigen expression. Tumor necrosis factor alpha (TNFα), which alone had no effect, synergistically enhanced this effect of IFNγ. MHC class II antigen expression was observed to be independent of cell cycle phase. Our results indicate that IFNγ alters immature crypt epithelial cell turnover and upregulates MHC class II expression. These alterations may be important in the pathogenesis of immune-mediated bowel disorders. J. Cell. Physiol. 176:120–126, 1998. © 1998 Wiley-Liss, Inc.     

Non-Circadian Periodicity in the Duration of the Cell Cycle and the G1 Phase in the Hindlimb Epidermis of the Anuran Tadpole,Rana Pipiens

Using the percentage labeled mitoses method, seven cell cycle determinations were initiated at 6-hr intervals over a 36-hr span in order to see if the cell cycle in the tadpole hindlimb epidermis varied with time or showed rhythmicity. There was a pattern of two long cell cycles followed by a shorter one. Total cell cycle length (Tc) and the length of the G₁ phase plus one-half of the mitotic time (TG₁+½M) fluctuated the most, although only TG₁+½M varied significantly with the Chi-square test. The proportion of TC spent in each phase was also calculated. Only TG₁+½M/Tc had statistically significant fluctuations with time.

Rhythmicity was analyzed by a computer program using the method of least squares for cosine curve fitting. Statistically significant ultradian rhythms of 18.4 hr in TC, 18.5 hr in TG₁+½M and 18.6 hr in TG₁+½M/TC and the length of the DNA synthetic phase/total cell cycle length (TS/TC) were found. Circadian rhythmicity was not observed. The acrophases of the ultradian rhythms of TC and TG₁+½M coincided, suggesting that the rhythm of TC was due mainly to variation in TG₁+½M. In the absence of significant variation in TS, the longest phase of the cell cycle, whenever G₁+½M was short, TS/TC increased, so that the 18.6 hr rhythm in TS/TC was also a result of the periodicity in TG₁+½M.     

THE EFFECT OF VINCRISTINE ON MOUSE JEJUNAL CRYPT CELLS OF DIFFERING CELL AGE: DOUBLE LABELLING AUTORADIOGRAPHIC STUDIES USING 3H- AND 14C-TdR

The mechanism of action of the alkaloid vincristine (VCR) has been investigated in vitro on HeLa cells in culture and in vivo on jejunal crypt cells of the mouse. The in vitro experiments with HeLa cells show that VCR affects not only mitotic but also interphase cells. The VCR-affected cells first continue their passage through the cell cycle undisturbed but after reaching mitosis they are arrested in metaphase. This agrees well with the results obtained by Madoc-Jones & Mauro (1968) and Madoc-Jones (1973) on synchronized cell cultures. Until now there has been no investigation of the mechanism of action of VCR in vivo. This is due to the absence of a suitable technique for synchronization in vivo. The present study is based on a method which permits the assessment of the VCR sensitivity as a function of the cell age without synchronization in the usual sense. The jejunal crypt epithelium of the normal mouse was double labelled with ³H- and ¹⁴C-thymidine (TdR) in such a way as to produce a narrow subpopulation of crypt cells with a maximum age difference of 1 hr. On autoradiographs these cells can be distinguished by their characteristic labelling from other cells. As this ‘pseudo’-synchronized subpopulation passes through the cycle the effect of VCR can be studied, i.e. one can analyse the effect in well-defined time intervals of the cycle. The results show that the effect of VCR is the same in vivo as in vitro. The crypt cells which are affected by VCR in interphase continue their passage through the cycle, but upon entering mitosis they are arrested in metaphase. VCR has, at the concentration used in the present study, no effect on the duration of the S and G₂ phases. The necrotic cells seen after VCR application are formed from arrested metaphases.     

ON THE EXISTENCE OF A Go-PHASE IN THE CELL CYCLE

The model is based on the assumption that the cell cycle contains a G_o-phase which cells leave randomly with a constant probability per unit time, γ. After leaving the G_o-phase, the cells enter the C-phase which ends with cell division. The C-phase and its constituent phases, the‘true’G₁-phase, the S-phase, the G₂-phase and mitosis are assumed to have constant durations of T, T₁T_s, T₂ and T_m, respectively. For renewal tissue it is assumed that the probability per unit time of being lost from the population is a constant for all cells irrespective of their position in the cycle. The labelled mitosis curve and labelling index for continuous labelling are derived in terms of γ, T, and T_s. The model generates labelled mitosis curves which damp quickly and reach a constant value of twice the initial labelling index, if the mean duration of the G_o-phase is sufficiently long. It is shown that the predicted labelled mitosis and continuous labelling curves agree reasonably well with the experimental curves for the hamster cheek pouch if T has a value of about 60 hr. Data are presented for the rat dorsal epidermis which support the assumption that there is a constant probability per unit time of a cell being released from the Go-phase.     

PROLIFERATION KINETICS OF AN EXPERIMENTAL ASCITES TUMOUR OF THE MOUSE

The cell cycles of an experimental ascitic tumour of the C3H mouse (NCTC 2472) were determined at various times after the intraperitoneal injection of 10⁶ cells. It was found that, contrary to results in solid NCTC 2472 tumours, obtained with the same NCTC cells, the duration of the cell cycle and its phases lengthened with the age of the tumour while the growth fraction remained relatively constant. G₁ was the first phase to lengthen, while later T_s and T_G2 increased also. The amount of DNA per cell was determined by cytospectrophotometry. This method provides data on the evolution during growth of the relative number of cells in each phase of the cell cycle.     

Interfering transbody-mediated Her2 gene silencing induces apoptosis by G0/G1 cell cycle arrest in Her2-overexpressing SK-BR-3 breast cancer cells

We previously isolated an interfering transbody, 4MH2, which penetrated the cytosol of living cells and preferentially hydrolyzed the target Her2 (ErbB2) mRNA, resulting in Her2 gene silencing followed by apoptotic cell death in Her2-overexpressing breast cancer cells. Here, we report the apoptotic cell death mechanism mediated by 4MH2-induced Her2 gene silencing in Her2-overexpressing SK-BR-3 breast cancer cells, in comparison with a small interfering RNA (siRNA) targeting Her2 mRNA (Her2₁₈-siRNA). 4MH2 induced G₀/G₁ cell cycle arrest to cause apoptotic cell death in SK-BR-3 cells by triggering specific signaling pathways associated with Her2 knockdown, including upregulation of G₀/G₁ cell cycle arrest-associated p21^Cip1 and p27^Kip1, downregulation of cyclin D1, inhibition of Akt phosphorylation, and downregulation of antiapoptotic Bcl-xL, which are comparable to those mediated by Her2₁₈-siRNA. Our results suggest that 4MH2-mediated Her2 gene silencing can trigger the downstream signaling pathways caused by Her2 downregulation, comparable to those mediated by the corresponding siRNA.     

Cell cycle arrest by prostaglandin A1 at the G1/S phase interface with up-regulation of oncogenes in S-49 cyc− cells

Our previous studies have implied that prostaglandins inhibit cell growth independent of cAMP. Recent reports, however, have suggested that prostaglandin arrest of the cell cycle may be mediated through protein kinase A. In this report, in order to eliminate the role of c-AMP in prostaglandin mediated cell cycle arrest, we use the-49 lymphoma variant (cyc^?) cells that lack adenylate cyclase activity. We demonstrate that dimethyl prostaglandin A₁ (dmPGA₁) inhibits DNA synthesis and cell growth in cyc^? cells. DNA synthesis is inhibited 42% by dmPGA₁ (50 μM) despite the fact that this cell line lacks cellular components needed for cAMP generation. The ability to decrease DNA synthesis depends upon the specific prostaglandin structure with the most effective form possessing the α,β unsaturated ketone ring. Dimethyl PGA₁ is most effective in inhibiting DNA synthesis in cyc^? cells, with prostaglandins PGE₁ and PGB₁ being less potent inhibitors of DNA synthesis. DmPGE₂ caused a significant stimulation of DNA synthesis. S-49 cyc^- variant cells exposed to (30–50 μm) dmPGA₁, arrested in the G₁ phase of the cell cycle within 24 h. This growth arrest was reversed when the prostaglandin was removed from the cultured cells; growth resumed within hours showing that this treatment is not toxic. The S-49 cyc^? cells were chosen not only for their lack of adenylate cyclase activity, but also because their cell cycle has been extensively studied and time requirements for G₁, S, G₂, and M phases are known. Within hours after prostaglandin removal the cells resume active DNA synthesis, and cell number doubles within 15 h suggesting rapid entry into S-phase DNA synthesis from the G₁ cell cycle block. The S-49 cyc^? cells are known to have a G₁/S boundary through M phase transition time of 14.8 h, making the location of the prostaglandin cell cycle arrest at or very near the G₁/S interface. The oncogenes, c-fos and c-myc which are normally expressed during G₁ in proliferating cells have a 2–3 fold enhanced expression in prostaglandin G₁ arrested cells. These data using the S-49 variants demonstrate that dmPGA₁ inhibits DNA synthesis and arrests the cell cycle independent of cAMP-mediated effects. The prostaglandin arrested cells maintain the gene expression of a G₁ synchronous cell which suggests a unique molecular mechanism for prostaglandin action in arresting cell growth. These properties indicate that this compound may be an effective tool to study molecular mechanisms of regulation of the cell cycle.     

Cell Synchrony Techniques. II. Analysis of Cell Progression Data

Abstract CHO cells which have been sorted by mitotic detachment, centrifugal elutriation and fluorescence activated cell sorting have been followed for up to 14 hr by flow cytometry to examine their progression characteristics. Mathematical modelling techniques were used to provide quantitative estimates of the cell-cycle parameters. Mitotic detachment gives an 11.2-hr cycle time with mean transit times T_G1, T_s and T_G2M equal to 3.2, 5.6 and 2.4 respectively. Cells prepared by central elutriation in an early G₁ state have a 14-hr cycle time with T_G1, T_s and T_G2M of 5.7, 6.0 and 2.3 hr. Populations prepared by centrifugal elutriation enriched in early S and late S and G₂M have transit times of 2.7, 5.9 and 1.6 hr and 4.9, 6.7 and 2.1 hr with cycle times of 11.2 and 13.2 hr respectively. Cell sorting for a G₁ population gives transit times of 9.8, 8.0 and 3.6 for an overall 21.4-hr cycle time.     

The effect of ethylene on root meristems of Pisum sativum and Zea mays

Summary Ethylene at a concentration of 100 l l^–1 causes a slight increase in the duration of the mitotic cycle in the primary root meristems of both Pisum sativum L. and Zea mays L. This is due to a lengthening of the G ₁ phase; other phases of the cycle are unaffected. Autoradiography and microdensitometry show that the rate of ³H-thymidine incorporation into nuclei of Pisum is maximal when about half the DNA has been replicated, and that ethylene has no effect upon this rate. Ethylene causes a reduction of the number of dividing cells in the root meristem, particularly in Pisum.Abbreviations Duration of the S phase, the G ₁ phase, the G ₂ phase of the mitotic cell cycle, respectively - T _C Duration of the complete mitotic cell cycle - QC Quiescent centre - LI, MI Labelling index, Mitotic index (i.e. fraction of the population labelled or in mitosis, respectively) - PF Proliferative fraction (i.e. fraction of the population making progress towards mitosis) - [³H]dT tritiated thymidine     

MATRIX SIMULATION OF DUODENAL CRYPT CELL KINETICS

Steady state crypt cell kinetics have been simulated using matrix algebra. The model crypt cell population is distributed through two proliferation compartments (P₁ and P₂) and a quiescent state (Q). Under steady state conditions half the daughter cells produced on completion of P₁ enter G₁ of P₂ and half enter G₁ of P₁. Both P₂ daughter cells enter Q. Cells in Q are non-dividing but retain the potential to divide. On completion of Q, cells lose the potential to divide and move up onto the villi. The model has been developed by simultaneously simulating the following biological data: (1) the per cent labeled mitosis (PML) curve, (2) the number of labeled cells per crypt as a function of time following an injection of ³H-thymidine, and (3) the total number of cells per crypt.     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

Cell proliferation in the subependymal layer of the adult mouse in vivo and in vitro

Pulse labelling experiments with [³H] thymidine (dT) and double labelling experiments with [³H]dT and bromodeoxyuridine (BrdUrd) were carried out on cells of the subependymal layer in the brain of adult normal mice in vivo, in vivo/in vitro and in vitro. The results should (i) lead to information about cell cycle parameters of these cells in the brain of adult mice, since these cells have been studied mostly in the rat brain up to now and (ii) answer the question whether results concerning cell proliferation obtained in vivo correspond with those from brain slices incubated in vitro with or without prelabelling in vivo. In vivo an LI of 20.2 ± 2.7% (x?± SEM) and T_s= 7.2 ± 0.7h were found. Furthermore, grain count halving experiments led to a surprisingly short cycle time (T_c) of 11.2–14.2 h. The longer T_c values (18–20 h) reported in the literature for subependymal cells in the rat brain seem to be due to evaluations of different areas around the lateral ventricle without considering the migrating behaviour of these cells which is quite different regionally. The in vitro studies (with or without prelabelling in vivo) showed a significantly reduced LI due to the fact that about 20% of the S phase cells, possibly lying in the middle of S, stopped further DNA synthesis after transfer to culture. This was shown by comparing the cell fluxes at the G₁/S and S/G₂ borders of in vivo vs. in vitro studies.     

Effect of staurosporine on MOLT-4 cell progression through G2 and on cytokinesis

Staurosporine (SSP) is an inhibitor of a variety of protein kinases with an especially high affinity towards protein kinase C. Whereas SSP has been shown to halt the cell cycle progression of various normal, nontransformed cell types in G₁, most virus transformed or tumor cells are unaffected in G₁ but arrest in G₂ phase. SSP has also been observed to increase the appearance of cells with higher DNA content, suggestive of endoreduplication, in cultures of tumor cells. Using multivariate flow cytometry (DNA content vs. expression of cyclin B, nucleolar p120 protein, or protein reactive with Ki-67 antibody) which makes it possible to discriminate cells with identical DNA content but at different phases of the cycle, we have studied the cell cycle progression of human lymphocytic leukemic MOLT-4 cells in the presence of 0.1 μM SSP.MOLT-4 cells did not arrest in G₁ or G₂ phase in the presence of the inhibitor. Rather, they failed to undergo cytokinesis, entering G₁ phase at higher DNA ploidy (tetraploidy; G_1T), and then progressed through S_T (rereplication) into G_2T and M_T. The rates of entrance to G₂ and G_2T were essentially identical, indicating that the rates of cell progression through S and S_T as well as through G₂ and G_2T, respectively, were similar. Cells entrance to mitosis and mitotic chromatin condensation were also similar at the diploid and tetraploid DNA content level and were unaffected by 0.1 μM SSP. No evidence of growth imbalance (altered protein or RNA to DNA ratio) was observed in the case of tetraploid cells. The data show that, in the case of MOLT-4 cells, all events associated with the chromosome or DNA cycle were unaffected by SSP; the only target of the inhibitor appears to be kinase(s) controlling cytokinesis. © 1994 Wiley-Liss, Inc.     

ANALYSIS OF THE GROWTH KINETICS OF A HUMAN LYMPHOMA CELL LINE

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 x 10⁴ - 5 x 10⁵ cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with ³H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. the average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. the generation time determined by PLM was 31.1 hr with a SD of 4.7 hr. Transit times for each stage were: T_G1= 10.6 hr, T_s= 9.9 hr, T_G2= 9.9 hr, and T_m= 0.7 hr. Repeated experiments using continuous labeling with ³H-TdR demonstrated a T_G2 of 6.3 hr. the longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give T_G1= 14.1 hr, T_s= 9.9 hr, T_G2 = 6.3 hr, and T_m= 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. the average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

Tumor-specific cell-cycle decoy by Salmonella typhimurium A1-R combined with tumor-selective cell-cycle trap by methioninase overcome tumor intrinsic chemoresistance as visualized by FUCCI imaging

We previously reported real-time monitoring of cell cycle dynamics of cancer cells throughout a live tumor intravitally using a fluorescence ubiquitination cell cycle indicator (FUCCI). Approximately 90% of cancer cells in the center and 80% of total cells of an established tumor are in G₀/G₁ phase. Longitudinal real-time FUCCI imaging demonstrated that cytotoxic agents killed only proliferating cancer cells at the surface and, in contrast, and had little effect on the quiescent cancer cells. Resistant quiescent cancer cells restarted cycling after the cessation of chemotherapy. Thus cytotoxic chemotherapy which targets cells in S/G₂/M, is mostly ineffective on solid tumors, but causes toxic side effects on tissues with high fractions of cycling cells, such as hair follicles, bone marrow and the intestinal lining. We have termed this phenomenon tumor intrinsic chemoresistance (TIC). We previously demonstrated that tumor-targeting Salmonella typhimurium A1-R (S. typhimurium A1-R) decoyed quiescent cancer cells in tumors to cycle from G₀/G₁ to S/G₂/M demonstrated by FUCCI imaging. We have also previously shown that when cancer cells were treated with recombinant methioninase (rMETase), the cancer cells were selectively trapped in S/G₂, shown by cell sorting as well as by FUCCI. In the present study, we show that sequential treatment of FUCCI-expressing stomach cancer MKN45 in vivo with S. typhimurium A1-R to decoy quiescent cancer cells to cycle, with subsequent rMETase to selectively trap the decoyed cancer cells in S/G₂ phase, followed by cisplatinum (CDDP) or paclitaxel (PTX) chemotherapy to kill the decoyed and trapped cancer cells completely prevented or regressed tumor growth. These results demonstrate the effectiveness of the praradigm of “decoy, trap and shoot” chemotherapy.