A biodistribution study of PEGylated PCL-based nanoparticles in C57BL/6 mice bearing B16/F10 melanoma.

One of the major drawbacks that limits the clinical application of nanoparticles is the lack of preliminary investigations related to their biocompatibility, biodegradability and biodistribution. In this work, biodegradable PEGylated polymer nanoparticles (NPs) have been synthesized by using macromonomers based on poly(ε-caprolaconte) oligomers. More in detail, NPs have been produced by adopting a surfactant-free semibatch emulsion polymerization process using PEG chains as a stabilizing agent. The NPs were also labeled with rhodamine B covalently bound to the NPs to quantitatively study their biodistribution in vivo. NPs were investigated in both in vitro and in vivo preclinical systems to study their biodistribution in mice bearing B16/F10 melanoma, as well as their biocompatibility and biodegradability. The NP concentration was evaluated in different tissues at several times after intravenous injection. The disappearance of the NPs from the plasma was biphasic, with distribution and elimination half-lives of 30 min and 15 h, respectively. NPs were retained in tumors and in filter organs for a long time, were still detectable after 7 d and maintained a steady concentration in the tumor for 120 h. 48 h after injection, 70 ± 15% of the inoculated NPs were excreted in the feces. The favorable tumor uptake, fast excretion and absence of cytotoxicity foster the further development of produced NPs as drug delivery carriers.

Synergistic antitumor activity of lapatinib and retinoids on a novel subtype of breast cancer with coamplification of ERBB2 and RARA.

All-trans retinoic acid (ATRA), the only clinically available cyto-differentiating agent, has potential for the therapy/chemoprevention of breast carcinoma. Given the heterogeneous nature of this tumor, a rational use of ATRA and derivatives (retinoids) in the clinic requires the identification of patients that would benefit from retinoid-based protocols. Here, we demonstrate that 23-32% of the human ERBB2(+) breast cancers show coamplification of retinoic acid receptor alpha (RARA), encoding the retinoic acid receptor, RARα. This represents a novel subtype of breast cancer characterized by remarkable sensitivity to ATRA and RARα agonists, regardless of positivity to the estrogen receptor, a known modulator of retinoid sensitivity. In estrogen-receptor-negative cellular models showing coamplification of ERBB2 and RARA, simultaneous targeting of the corresponding gene products with combinations of lapatinib and ATRA causes synergistic growth inhibition, cyto-differentiation and apoptosis. This provides proof-of-principle that coamplification of ERBB2 and RARA can be exploited for the stratified and targeted therapy of a novel subtype of breast cancer patients, with an approach characterized by tumor cell selectivity and low predicted toxicity. The available cellular models were exploited to define the molecular mechanisms underlying the antitumor activity of combinations between lapatinib and ATRA. Global gene expression and functional approaches provide evidence for three components of the antiproliferative/apoptotic responses triggered by lapatinib+ATRA. Induction of the retinoid-dependent RARRES3 protein by ATRA stabilizes the effect of lapatinib inhibiting ERBB2 phosphorylation. Upregulation and activation of the transcription factor FOXO3A integrates ATRA-dependent transcriptional and lapatinib-dependent posttranscriptional signals, controlling the levels of effector proteins like the antiapoptotic factor, BIRC5. Stimulation of the TGFß pathway by ATRA mediates other components of the apoptotic process set in motion by simultaneous targeting of ERBB2 and RARα.

Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach.

OBJECTIVES: Trabectedin (ET-743, Yondelis) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)-rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug-induced cell cycle perturbations. MATERIALS AND METHODS: An isogenic NER-proficient cellular system (CHO-AA8) and a NER-deficient one (CHO-UV-96), lacking functional ERCC-1, were studied. Flow cytometric assays showed progressive accumulation of cells in G2 + M phase in NER-proficient but not in NER-deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. RESULTS: Cells exposed to trabectedin during G1 and G2 + M first experienced a G1 block, while those exposed in S phase were delayed in S and G2 + M phases but eventually divided. In the presence of functional NER, exit from the G1 block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G2 + M phase. This G2 + M processing of trabectedin-induced damage in NER-proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. CONCLUSIONS: This might be due either to important damage left unrepaired by previous G1 repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV-96 cells repair mechanisms other than NER are activated both in G1 and G2 + M phases.

The combination of yondelis and cisplatin is synergistic against human tumor xenografts.

Yondelis (trabectidin, ET-743) is a marine natural product that has shown activity both in preclinical systems and in human malignancies such as soft tissue sarcoma and ovarian cancers that are resistant to previous chemotherapies. Molecular pharmacological studies indicated that Yondelis interacts with DNA and DNA repair systems in a way that is different from Cisplatin (DDP). The current study was designed to investigate the effects of the combination of Yondelis and DDP in human cancer cell lines and in xenografts derived from different tumours. The in vitro studies performed in human TE-671 rhabdomyosarcoma, Igrov-1 and 1A9 human ovarian carcinoma cell lines showed additive effects or slight synergism. Several human tumour xenografts, such as TE-671 rhabdomyosarcoma, SK-N-DX neuroblastoma, FADU head and neck, LX-1 non-small cell lung cancer (NSCLC), H-187 melanoma and SKOV HOC 8 ovarian carcinoma, showed an antitumour effect for the combination that was greater than that of each drug when given as a single agent. No consistent changes in the activity were observed if Yondelis and DDP were given 1 h apart in sequence or simultaneously. An orthotopically transplanted human ovarian cancer HOC 8 growing in the peritoneal cavity of nude mice was used that is insensitive to Yondelis alone and only moderately sensitive to DDP alone. The combination of the two drugs produced a dramatic increase of survival lasting several months. In conclusion, the combination of Yondelis and DDP is synergistic in vivo (i.e. the antitumour effect is greater than that of each drug used as a single agent at the maximum tolerated dose (MTD)) in different human tumour xenografts. The two drugs can be combined at the MTD of each drug, thus indicating there are no overlapping toxicities. These results provide a rationale for testing the combination of Yondelis and DDP in the clinic.

Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine.

Aplidine, dehydrodidemnin B, is a marine depsipeptide isolated from the Mediterranean tunicate Aplidium albicans currently in phase II clinical trial. In human Molt-4 leukaemia cells Aplidine was found to be cytotoxic at nanomolar concentrations and to induce both a G(1) arrest and a G(2) blockade. The drug-induced cell cycle perturbations and subsequent cell death do not appear to be related to macromolecular synthesis (protein, RNA, DNA) since the effects occur at concentrations (e.g. 10 nM) in which macromolecule synthesis was not markedly affected. Ten nM Aplidine for 1 h inhibited ornithine decarboxylase activity, with a subsequently strong decrease in putrescine levels. This finding has questionable relevance since addition of putrescine did not significantly reduce the cell cycle perturbations or the cytotoxicity of Aplidine. The cell cycle perturbations caused by Aplidine were also not due to an effect on the cyclin-dependent kinases. Although the mechanism of action of Aplidine is still unclear, the cell cycle phase perturbations and the rapid induction of apoptosis in Molt-4 cells appear to be due to a mechanism different from that of known anticancer drugs.

Solar UV radiation: differential effectiveness of UVB subcomponents in causing cell death, micronucleus induction and delayed expression of heritable damage in human hybrid cells.

PURPOSE: To determine the effectiveness of two UV spectra with different UVB components for cell kill and micronucleus induction in irradiated human HeLaxskin fibroblast (CGL1) hybrid cells and their progeny. To determine the presence of reactive oxygen species (ROS) in the progeny of the irradiated cells at various post-irradiation times and their relationship with induced delayed biological effects. MATERIAL AND METHODS: A commercial solar ultraviolet simulator was used. Two different filters were employed: the first transmitted radiation with lambda>284nm and the second radiation with lambda>293nm. The resulting spectra have different UVB components (lambda between 284 and 320nm, 19 W/m(2), and between 293 and 320nm, 13 W/m(2)) and the same UVA component (lambda between 320 and 400nm, 135 W/m(2)). CGL1 cells were irradiated with various doses. Clonogenic survival and micronucleus formation were scored in the irradiated cells and their progeny. ROS were detected by incubation of cultures at various post-irradiation times with dichlorodihydrofluorescein diacetate followed by flow cytometric measurement of the final product, dichlorofluorescein. RESULTS: The biological effectiveness of the lambda>284nm spectrum was higher by a factor of 3 compared to the lambda>293nm spectrum for cell kill, and by a factor of 5 for micronucleus induction. No delayed cell death or micronucleus formation was found in the progeny of cells exposed to lambda>293nm, while a large and dose-dependent effect was found in the progeny of cells exposed to lambda>284nm for both of these endpoints. ROS levels above those in unirradiated controls were found only in the progeny of cells exposed to the lambda>284nm spectrum. CONCLUSIONS: The spectrum with lambda>284nm was more effective than that with lambda>293nm for induction of cell kill and micronucleus formation in the directly irradiated cells as well as induction of delayed effects in the progeny in the form of delayed reproductive death and micronucleus formation. The presence of ROS in the progeny of the irradiated cells may be the cause of the delayed effects.

Cell cycle effects of gemcitabine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, or dFdC) is a promising anticancer agent with demonstrated clinical activity in solid tumours currently undergoing clinical trials. Despite extensive studies on the biochemical mechanism of action, cell cycle perturbations induced by dFdC have not yet been thoroughly investigated, apart from the expected inhibition of DNA synthesis. The aim of our study was to clarify whether cell population kinetics is a vital factor in the cytotoxicity of dFdC in single or repeated treatments and in the dFdC-cisplatin combination. Ovarian cancer cells growing in vitro were treated with dFdC for 1 hr in a range of concentrations from 10 nM to 10 microM. Cell kinetics was investigated by DNA-bromodeoxyuridine flow cytometry, using different experimental protocols to measure either the time course of DNA-synthesis inhibition or the fate of cells in G(1), S or G(2)M at the time of dFdC treatment or 24 hr later. A modified sulforhodamine B test was used to assess the growth inhibition caused by dFdC given alone or with cisplatin. Although dFdC promptly inhibited DNA synthesis, cytotoxicity on proliferating cells was not specific for cells initially in the S phase. DNA synthesis was restored after a G(1) block of variable, dose-dependent length, but recycling cells were intercepted at the subsequent checkpoints, resulting in delays in the G(2)M and G(1) phases. The activity of repeated treatment with dFdC + dFdC or dFdC + cisplatin was highly dependent on the interval length between them. These results suggest that the kinetics of cell recycling from a first dFdC treatment strongly affects the outcome of a second treatment with either dFdC itself or cisplatin.

Desynchronization rate in cell populations: mathematical modeling and experimental data.

We characterize the kinetics of two cancer cell lines: IGROV1 (ovarian carcinoma) and MOLT4 (leukemia). By means of flow cytometry, we selected two populations from exponentially growing in vitro cell lines, depending on the cells' DNA synthesis activity during a preceding labeling period. For these populations we determined the time course of the percentages of cells in different phases of the cycles, sampling every 3 hr for 60 hr. Initially, semi-synchronous populations quickly converged to a stable age distribution, which is typical of the cell line (at equilibrium); this desynchronization reflects the intercell variability in cell cycle duration. By matching these experimental observations to mathematical modelling, we related the convergence rate toward the asymptotic distribution (R) and the period of the phase-percentage oscillations (T), to the mean cell cycle duration and its coefficient of variation. We give two formulas involving the above-mentioned parameters. Since T and R can be drawn by fitting our data to an asymptotic formula obtained from the model, we can estimate the other two kinetic parameters. IGROV1 cells have a shorter mean cell cycle time, but higher intercell variability than the leukemia line, which takes longer to lose synchrony.

Measuring the complexity of cell cycle arrest and killing of drugs: kinetics of phase-specific effects induced by taxol.

BACKGROUND: Paclitaxel (Taxol) is known to act mainly in mitosis, interfering with microtubule dynamics, but effects on the other cells cycle phases have been reported also. However, a comparative picture of perturbation and killing in the G(1), S and G(2)M phases after drug treatment is lacking. The approach developed by our group tackles the problem of the complexity of cell cycle effects with the aid of a computer program simulating cell cycle progression and new quantities measuring cell-cycle arrest and death. METHODS: The program generates data that were compared with those given by absolute cell counts and by different flow cytometry techniques, enabling us to follow the fate of G(1) and G(2)M blocked cells either re-entering the cycle or dying, distinguishing cytostatic and cytotoxic effects. Apoptosis was analyzed in order to refine the description of cytotoxic effects. RESULTS: We estimated the number of blocked and dead cells after short-term Taxol treatments in a range of concentrations and post-drug incubation times. G(2)M block was immediately active at low concentrations but was reversible, becoming irreversible only at the highest concentrations. G(1)block became active later, allowing cell cycle progression of cells initially in G(1), but was still active 48 h post-treatment, at intermediate concentrations. S-phase delay was detected after 24 h. The death rate was much higher within G(1)than G(2)M blocked cells. CONCLUSIONS: Our analysis unraveled the complexity of cell cycle effects of the drug, and revealed the activity of G(1) checkpoint, hidden by a prompter but less cytotoxic G(2)M block.

Dose estimate of exposure to radioisotopes in molecular and cellular biology.

A method for prospectively evaluating the annual equivalent doses and effective dose to biomedical researchers working with unsealed radioisotopes, and their classification, is presented here. Simplified formulae relate occupational data to a reasonable overestimate of the annual effective dose, and the equivalent doses to the hands and to the skin. The procedure, up to the classification of personnel and laboratories, can be made fully automatic, using a common spreadsheet on a personal computer. The method is based on occupational data, accounting for the amounts of each radioisotope used by a researcher, the time of exposure and the overall amounts employed in the laboratories where experiments are performed. The former data serve to forecast a contribution to the dose arising from a researcher's own work, the latter to a forecast of an 'environmental' contribution deriving simply from the presence in a laboratory where other people are working with radioisotopes. The estimates of the doses due to one's own radioisotope handling and to 'environment' were corrected for accidental exposure, considered as a linear function of the manipulated activity or of the time spent in the laboratories respectively, and summed up to give the effective dose. The effective dose associated with some common experiments in molecular and cellular biology is pre-evaluated by this method.

The antitumor efficacy of cytotoxic drugs is potentiated by treatment with PNU 145156E, a growth-factor-complexing molecule.

PNU 145156E (formerly FCE 26644) is a noncytotoxic molecule whose antitumor activity is exerted through the formation of a reversible complex with growth/angiogenic factors, thus inhibiting their induction of angiogenesis. We studied in vitro and in vivo the activity of PNU145156E in combination with the four cytotoxic drugs doxorubicin, cyclophosphamide, methoxymorpholinyldoxorubicin (MMDX, FCE 23762, PNU152243), and 9-aminocamptothecin against M5076 murine reticulosarcoma. In vitro, PNU 145156E did not modify the cytotoxicity of the four drugs or the cell-cycle block induced by doxorubicin. In vivo, at the optimal dose of each compound, the antitumor activity was significantly increased in all combinations, with no associated increase in general toxicity being observed. In healthy mice treated with cyclophosphamide or doxorubicin the association with PNU 145156E did not enhance the myelotoxic effect induced by the two cytotoxics. These results indicate that two drugs affecting solid tumor growth through two different mechanisms-growth factor blockage and cell proliferation can be combined, resulting in increased antitumor efficacy with no additive toxicity.

Characterization of cyclin B1 expression in human cancer cell lines by a new three-parameter BrdUrd/cyclin B1/DNA analysis.

Flow cytometric cyclin expression/DNA content analysis, now commonly used, provides useful information on the mechanisms regulating cell cycle progression. However, this biparametric analysis does not make a clear-cut distinction between G1 and S-early or between S-late and G2M phase cells. This paper proposes a new three-parameter flow cytometric method with which to determine cyclin B1 levels in single cells in different cell cycle phases by coupling bromodeoxyuridine (BrdUrd) immunodetection and DNA content. DNA denaturation by HCl did not alter the level of cyclin B1. Differences in cyclin B1 expression were observed in seven human cancer cell lines of different origin. The percentage of cyclin B1-positive cells and the cyclin B1 content per cell indicated different patterns. In some cases cyclin B1 accumulation preceded the G2M checkpoint, at which its content usually started to rise. Using available easily reproducible techniques, this flow cytometric approach gives details of intracellular variability in cyclin expression.

From flow cytometric BrdUrd data to cell population growth and doubling time.

We describe a direct way to use flow cytometric data for measuring the growth curve of a cell population. The starting point is analysis of the intrinsic informative content of the time course, after bromodeoxyuridine (BrdUrd) labeling, of the percentages of cells detected within four windows of biparametric BrdUrd-DNA histograms. We did not introduce a particular cell cycle model or use the hypothesis of exponential growth. We obtained a simple formal proof of the existence of four independent formulae connecting the flow cytometric data and the relative growth curve of the cell population. The formulae were then challenged in a number of simulated kinetic scenarios, moving away from their expected limits of validity. The results suggest additional uses of the formulae and a way of estimating cell-cycle-phase durations. Considering exponential growth in the presence of cell loss, the formulae were used to estimate the potential doubling time from a single flow cytometric measure vs. other procedures that additionally require an estimate of the duration of the phase S. The theoretical precision of the procedures may differ depending on how cell loss occurs.

Cell loss and the concept of potential doubling time.

The in vivo infusion of Bromodeoxyuridine (BrdUrd), followed by delayed biopsy and bivariate DNA-BrdUrd flow cytometry, allows the potential doubling time (Tpot) of human tumors to be estimated. According to Steel, the mathematical definition of Tpot is Tpot = ln 2/Kp, where Kp is the rate constant of cell production. All the operative formulas which allow the estimation of Tpot from flow cytometric data derive from this definition. Most authors, however, identify the potential doubling time as the doubling time that the same cell population would exhibit if cell loss were removed. We denote here as T(d)noloss this quantity. Although these two definitions are equivalent in the case of uniform random cell loss, we show, in the framework of Steel's theory of growing cell populations, that Tpot and T(d)noloss become distinct kinetic quantities when cell loss is not uniform, i.e., when loss differently affects the quiescent and the proliferative compartment. We discuss the validity of the two formulas currently used for the calculation of Tpot, one based on LI and the other on the v-function, in conditions of non-uniform cell loss. Moreover, we propose two formulas for the estimation of the cycle time T(C), which require, in addition to T(S) and LI, that a measure of the growth fraction be available.

Hematopoietic toxicity and cell cycle perturbations induced by new DNA minor groove-alkylating agents.

Some new alkylating agents which bind to the minor groove of DNA and have sequence-specific patterns of alkylation have shown anti-neoplastic activity in pre-clinical systems. Two of them, carzelesin and tallimustine, are now in phase II. Considering the severe dose-limiting bone marrow toxicity of both these drugs in clinical use, it was of interest to investigate the mechanism of their myelotoxicity in a detailed pre-clinical study and compare it with a conventional alkylating agent, such as melphalan. The origin and progression of the myelotoxicity of carzelesin, tallimustine and melphalan were investigated comparatively in mice, combining data on bone marrow and peripheral blood cellularity with data on the proliferative activity of bone marrow cells, obtained by in vivo administration of bromodeoxyuridine. Significant differences were found between the hematopoietic response to the 3 drugs, though all caused severe leukopenia. Carzelesin induced a short-term increase in myeloid proliferative activity, which prevented the high leukocytopenia on day 3 observed with the other drugs. However, when this effect was exhausted, a second nadir was seen in peripheral blood, with a new wave of cell proliferation of all lineages in the bone marrow. Reconstruction of the lymphoid lineage was slow for all 3 drugs but particularly difficult with high-dose tallimustine. In general, the hematopoietic system response to tallimustine was dampened, with no overshoots, suggesting either lasting effects or extensive cytotoxicity from the early to late precursors of all lineages.

Quality control study of the Italian Group of Cytometry on flow cytometry DNA content measurements: II. Factors affecting inter- and intralaboratory variability.

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.

Combination of the new minor groove alkylator tallimustine and melphalan.

The benzoyl nitrogen mustard derivative of distamycin A, tallimustine, belongs to a new class of alkylating agents, known as DNA minor groove alkylating agents. It alkylates adenine N3 with high sequence specificity, causing no alkylation of guanine N7, the main site of alkylation of clinically used nitrogen mustards such as L-PAM. The present study investigated the in vivo antitumour activity of a combination of tallimustine and melphalan (L-PAM). Two murine tumours were used: i.p. (intraperitoneally) transplanted L1210 leukaemia and i.m. (intramuscularly) transplanted M5076 ovarian reticulum cell sarcoma (M5). In L1210, which is only marginally sensitive to tallimustine, the combination of tallimustine 3 mg/kg i.p. with L-PAM 10 mg/kg i.p. was as effective as 20 mg/kg L-PAM, which is the maximum tolerated dose. In M5, which is sensitive to both drugs, the combination was superior to either drug alone. The results suggest that the combination of tallimustine and L-PAM--or possibly in general, minor groove alkylators and major groove alkylators--may be therapeutically advantageous and therefore should be investigated clinically.

Flow cytometric detection of glutathione S-transferase isoenzymes by quantitative immunofluorescence under nonsaturating conditions.

The glutathione (GSH)-glutathione S-transferase (GST) detoxification system is an important element in cellular defence against injurious agents and anticancer drugs. GST isoenzymes may represent biochemical markers of neoplastic transformation, and, possibly, drug resistance is associated with altered GST-isoenzyme levels. The ability to measure GST-isoenzymes in cell populations would be useful for several biological and clinical applications. We have developed an immunofluorescence flow cytometric method for the simultaneous detection of different GST-isoenzymes and of DNA in fixed cells. Due to the impossibility of working under saturating conditions for the anti-GST antibody, a normalizing procedure was developed to permit quantitative analysis of single cells labelled with the anti-GST antibody at high dilution. A theoretical model and experimental data supported the use of this procedure. The method proposed is general and could be applied to other antibodies in order to obtain quantitative data outside saturating conditions. The method was challenged in different applications in order to compare it with other classical techniques. First, we characterized sublines resistant to different anticancer drugs with respect to variations of GST isotypes. In a second application, we studied the intercellular heterogeneity of GST content in mouse renal cells. In addition, GST was determined in aneuploid cells from solid tumor biopsies by separation from diploid cells on the basis of DNA content. Finally, GST distribution during cell-cycle progression was studied in two different cell lines by the biparametric analysis of GST/DNA.

Increasing 1-beta-D-arabinofuranosylcytosine efficacy by scheduled dosing intervals based on direct measurements of bone marrow cell kinetics.

The therapeutic efficacy of cell cycle phase-specific drugs can be improved by repeated administrations, the dosing interval being related to the cell cycle time of the susceptible normal host tissue. Kinetic measurements of bone marrow cell proliferation, with bromodeoxyuridine labeling and flow cytometry analysis, were used to determine the optimal dosing intervals of 1-beta-D-arabinofuranosylcytosine for minimizing bone marrow cell damage in mice. The results showed that cells surviving a single dose 1-beta-D-arabinofuranosylcytosine treatment remained temporarily blocked at the G1-S boundary, and upon release from the block the cells crossed through S phase in a nearly synchronized way. The optimal spacing of repeated treatments, evaluated by measurements of the drug-induced transit times through the different cell cycle phases, equaled the bone marrow cell cycle time following treatment. Repeated 1-beta-D-arabinofuranosylcytosine injections according to this protocol markedly diminished drug toxicity in C3H mice, as compared to protocols of other time intervals. A therapeutic schedule based on these measurements was highly effective in lymphoma-bearing mice: the designed protocol of dosing intervals significantly delayed tumor growth whereas other intervals were highly toxic.