Signaling via the anti-CD30 mAb SGN-30 sensitizes Hodgkin's disease cells to conventional chemotherapeutics.

SGN-30, a monoclonal antibody with activity against CD30+ malignancies, is currently in phase II clinical evaluation for treatment of Hodgkin's disease (HD) and anaplastic large cell lymphoma. The mechanisms underlying SGN-30's antitumor activity were investigated using cDNA array of L540 cells. SGN-30 treatment activated NF-kappaB and modulation of several messages including the growth regulator p21WAF1/CIP1 (p21) and cellular adhesion marker ICAM-1. p21 protein levels increased coincident with growth arrest and Annexin V/PI staining in treated HD cells. To determine if SGN-30-induced growth arrest would sensitize tumor cells to chemotherapeutics used against HD, L540cy and L428 cells were exposed to SGN-30 in combination with a panel of cytotoxic agents and resultant interactions quantified by the Combination Effects Method. Interactions between SGN-30 and all cytotoxic agents examined were additive or better. These in vitro data translated to increased efficacy of SGN-30 and bleomycin against L540cy tumor xenografts. In addition to direct cell killing, SGN-30 affects growth arrest and drug sensitization through growth regulating and proapoptotic machinery. Importantly, these data suggest that SGN-30 can enhance the efficacy of standard chemotherapies used to treat patients with CD30+ malignancies.

Identification and activities of human carboxylesterases for the activation of CPT-11, a clinically approved anticancer drug.

CPT-11 is a clinically approved anticancer drug used for the treatment of advanced colorectal cancer. Upon administration, the carbamate side chain of the drug is hydrolyzed, resulting in the release of SN-38, an agent that has approximately 1000-fold increased cytotoxic activity. Since only a very small percentage of the injected dose of CPT-11 is converted to SN-38, there is a significant opportunity to improve its therapeutic efficacy and to diminish its systemic toxicity by selectively activating the drug within tumor sites. We envisioned that a mAb-human enzyme conjugate for CPT-11 activation would be of interest, particularly since the conjugate would likely be minimally immunogenic, and the prodrug is clinically approved. Toward this end, it was necessary to identify the most active human enzyme that could convert CPT-11 to SN-38. We isolated enzymes from human liver microsomes based on their abilities to effect the conversion and identified human carboxylesterase 2 (hCE-2) as having the greatest specific activity. hCE-2 was 26-fold more active than human carboxylesterase 1 and was 65% as active as rabbit liver carboxylesterase, the most active CPT-11 hydrolyzing enzyme known. The anti-p97 mAb 96.5 was linked to hCE-2, forming a conjugate that could bind to antigen-positive cancer cells and convert CPT-11 to SN-38. Cytotoxicity assays established that the conjugate led to the generation of active drug, but the kinetics of prodrug activation (48 pmol x min(-1) x mg(-1) was insufficient for immunologically specific prodrug activation. These results confirm the importance of hCE-2 for CPT-11 activation and underscore the importance of enzyme kinetics for selective prodrug activation.

Selective tumor sensitization to taxanes with the mAb-drug conjugate cBR96-doxorubicin.

The chimeric monoclonal antibody cBR96 conjugated to doxorubicin (cBR96-Dox) is selectively internalized by a wide variety of human carcinomas expressing an extended form of Lewis Y antigen (Le(y)). Endocytosis is followed by cleavage and release of free doxorubicin from the endocytic vesicles and subsequent cytotoxicity. Combination studies with standard anti-cancer agents, undertaken to further increase the potency of this targeted therapy, identified significant synergistic anti-tumor activity of cBR96-Dox and either of the taxanes paclitaxel or docetaxel. Treatment with cBR96-Dox 24 hr prior to paclitaxel resulted in a steady increase in the percentage of G(2) tumor cells and corresponding increase in sensitivity to taxanes. Cell cycle analysis indicated the cBR96-delivered doxorubicin was most effective against S-phase cells, yet cells exposed to even subtoxic levels progressed to and arrested in G(2), at a point of high sensitivity to the anti-tubulin agent paclitaxel. The synergy obtained by staged combination of cBR96-Dox and paclitaxel in vitro was reflected in significant anti-tumor efficacy in vivo against xenograft models of human lung and breast tumors that could not be achieved by either agent alone. The staged combination elicited significant or complete regressions of established human Le(y)-positive tumor xenografts using significantly reduced drug levels. Taken together, these data demonstrate a mechanistic approach to the selective elimination of Le(y)-positive tumors by using targeted doxorubicin followed by taxane treatment.

Oncostatin M in the anti-inflammatory response.

Oncostatin M (OM) is a pleiotropic cytokine of the interleukin 6 family, whose in vivo properties and physiological function remain in dispute and poorly defined. These in vivo studies strongly suggest that OM is anabolic, promoting wound healing and bone formation, and anti-inflammatory. In models of inflammation OM is produced late in the cytokine response and protects from lipopolysaccharide (LPS)-induced toxicities, promoting the re-establishment of homoeostasis by cooperating with proinflammatory cytokines and acute phase molecules to alter and attenuate the inflammatory response. Administration of OM inhibited bacterial LPS-induced production of tumour necrosis factor alpha and septic lethality in a dose dependent manner. Consistent with these findings, in animal models of chronic inflammatory disease OM potently suppressed inflammation and tissue destruction in murine models of rheumatoid arthritis and multiple sclerosis. T cell function and antibody production were not impaired by OM treatment. Taken together, these data indicate that the activities of this cytokine in vivo are anti-inflammatory without concordant immunosuppression.

Centrifugal elutriation to obtain synchronous populations of cells.

Counterflow centrifugal elutriation is a noninvasive method for separating large numbers of cells on the basis of their size and mass. For mammalian cells, this method is useful for separating mixed populations of cells, in particular cells at different stages of the cell division cycle without perturbing cell metabolism or using synchronizing agents. This unit describes a method for separating 2 x 10(8) cells using the standard JE-6B rotor or larger numbers of cells in the JE-5.0 rotor. To verify the purity and to characterize the cell cycle positions of cells in the elutriated populations, the unit includes protocols for measuring nascent DNA synthesis by [(3)H]thymidine incorporation and for detecting DNA synthesis and content by propidium iodide flow cytometry alone or in combination with bromodeoxyuridine incorporation.

Agonistic properties and in vivo antitumor activity of the anti-CD40 antibody SGN-14.

Ligation of CD40 is essential for primary B-cell activation and expansion and yet has suppressive or apoptotic effects on some CD40-expressing neoplasia. SGN-14 is a monoclonal antibody that binds to the human CD40 receptor. Here we report that SGN-14, in the presence of interleukin 4, provided a modest level of stimulation of peripheral blood B cells, as measured by proliferation. Stimulation was greatly enhanced in the presence of nonproliferating CD40 ligand-expressing cells. The enhanced agonistic activity could be attributed to a dose-dependent increase in CD40L binding to CD40 in the presence of SGN-14. In contrast to its proliferative effect on primary B cells, SGN-14 inhibited the growth of B-cell-derived tumor lines in vitro, and this growth inhibition was enhanced in the presence of CD40L-expressing cells. In vivo, SGN-14 showed significant antitumor activity in treating human B-cell lymphoma and multiple myeloma xenografted severe combined immunodeficient mice. Antitumor activity was not diminished by blunting murine natural killer activity, suggesting that CD40 ligation contributes to the antitumor efficacy of SGN-14. On the basis of these activities, SGN-14 is being pursued for therapeutic use in treating patients with CD40-expressing hematological malignancies.

Effect of flap modifications on human FEN1 cleavage.

The flap endonuclease, FEN1, plays a critical role in DNA replication and repair. Human FEN1 exhibits both a 5' to 3' exonucleolytic and a structure-specific endonucleolytic activity. On primer-template substrates containing an unannealed 5'-tail, or flap structure, FEN1 employs a unique mechanism to cleave at the point of annealing, releasing the 5'-tail intact. FEN1 appears to track along the full length of the flap from the 5'-end to the point of cleavage. Substrates containing structural modifications to the flap have been used to explore the mechanism of tracking. To determine whether the nuclease must recognize a succession of nucleotides on the flap, chemical linkers were used to replace an interior nucleotide. The nuclease could readily traverse this site. The footprint of the nuclease at the time of cleavage does not extend beyond 25 nucleotides on the flap. Eleven-nucleotide branches attached to the flap beyond the footprinted region do not prevent cleavage. Single- or double-thymine dimers also allow cleavage. cis-Platinum adducts outside the protected region are moderately inhibitory. Platinum-modified branch structures are completely inert to cleavage. These results show that some flap modifications can prevent or inhibit tracking, but the tracking mechanism tolerates a variety of flap modifications. FEN1 has a flexible loop structure through which the flap has been proposed to thread. However, efficient cleavage of branched structures is inconsistent with threading the flap through a hole in the protein.

Re-entry into the cell cycle: a mechanism for neurodegeneration in Alzheimer disease.

Several recent findings demonstrated increased expression of cell cycle-related proteins in the degenerating neurons found in Alzheimer disease. We hypothesize that this apparent attempt to re-enter the cell cycle is a neuronal response to external growth stimuli that leads to an abortive re-entry into the cell cycle. However, since neurons of adults apparently lack the capacity both to divide in vivo and in vitro, it is possible that they lack the components necessary to complete the cell division process. Nonetheless, the importance of these findings is that they provide an explanation for the increased phosphorylation of cytoskeletal proteins such as tau and neurofilaments that represent the most striking intracellular changes in the disease. Further, it is our contention that inappropriate reentry into the cell cycle and interrupted mitotic processes are significant factors not only in the cytoskeletal pathology but also in the neuronal degeneration that characterizes the pathology of Alzheimer disease.

Regulation of inflammatory responses by oncostatin M.

Oncostatin M (OM) is a pleiotropic cytokine produced late in the activation cycle of T cells and macrophages. In vitro it shares properties with related proteins of the IL-6 family of cytokines; however, its in vivo properties and physiological function are as yet ill defined. We show that administration of OM inhibited bacterial LPS-induced production of TNF-alpha and lethality in a dose-dependent manner. Consistent with these findings, OM potently suppressed inflammation and tissue destruction in murine models of rheumatoid arthritis and multiple sclerosis. T cell function and Ab production were not impaired by OM treatment. Taken together these data indicate the activities of this cytokine in vivo are antiinflammatory without concordant immunosuppression.

Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer's disease.

In this study, we demonstrate that two important regulators of the cell cycle, cyclin-dependent kinase-4 and its inhibitor p16, are increased in the brains of cases of Alzheimer's disease patients compared with age-matched controls. Both proteins are increased in the pyramidal neurons of the hippocampus, including those neurons containing neurofibrillary tangles and granulovacuolar degeneration. As p16 is not normally found in terminally differentiated neurons, it seems paradoxical that it is increased in Alzheimer's disease unless it is responding to increases in cyclin-dependent kinase-4 or other cell cycle regulators. Induction of the latter, a protein that signals re-entry and progression through the cell cycle, may itself be the consequence of alpha response to a growth stimulus. Re-entry into the cell cycle is likely deleterious in terminally differentiated neurons and may contribute to the biochemical abnormalities, such as oxidative stress and hyperphosphorylated tau protein, as well as the neuronal degeneration characteristic of the pathology of Alzheimer's disease.

Cyclin G2 is up-regulated during growth inhibition and B cell antigen receptor-mediated cell cycle arrest.

Human cyclin G2 together with its closest homolog cyclin G1 defines a novel family of cyclins (Horne, M. C., Goolsby, G. L., Donaldson, K. L., Tran, D., Neubauer, M., and Wahl, A. F. (1996) J. Biol. Chem. 271, 6050-6061). Cyclin G2 is highly expressed in the immune system where immunologic tolerance subjects self-reactive lymphocytes to negative selection and clonal deletion via apoptosis. Here we investigated the effect of growth inhibitory signals on cyclin G2 mRNA abundance in different maturation stage-specific murine B cell lines. Upon treatment of wild-type and p53 null B cell lines with the negative growth factor, transforming growth factor beta1, or the growth inhibitory corticosteroid dexamethasone, cyclin G2 mRNA levels were increased in a time-dependent manner 5-14-fold over control cell levels. Unstimulated immature B cell lines (WEHI-231 and CH31) and unstimulated or IgM B cell receptor (BCR) -stimulated mature B cell lines (BAL-17 and CH12) rapidly proliferate and express low levels of cyclin G2 mRNA. In contrast, BCR-stimulated immature B cell lines undergo growth arrest and coincidentally exhibit an approximately 10-fold increase in cyclin G2 transcripts and a decrease in cyclin D2 message. Costimulation of WEHI-231 and CH31 cells with calcium ionophores and protein kinase C agonists partially mimics anti-IgM stimulation and elicits a strong up-regulation of cyclin G2 mRNA and down-regulation of cyclin D2 mRNA. Signaling mutants of WEHI-231 that are deficient in the phosphoinositide signaling pathway and consequently resistant to the BCR stimulus-induced growth arrest did not display a significant increase in cyclin G2 or decrease in cyclin D2 mRNAs when challenged with anti-IgM antibodies. The two polyclonal activators lipopolysaccharide and soluble gp39, which inhibit the growth arrest response of immature B cells, suppressed cyclin G2 mRNA expression induced by BCR stimulation. These results suggest that in murine B cells responding to growth inhibitory stimuli cyclin G2 may be a key negative regulator of cell cycle progression.

Separation by counterflow centrifugal elutriation and analysis of T- and B-lymphocytic cell lines in progressive stages of cell division cycle.

The method of counterflow centrifugal elutriation (CCE) facilitates the non-invasive separation of proliferating cells into the progressive stages of the cell division cycle. We present here detailed protocols for the separation of primary lymphocytes and lymphocytic cell lines including Jurkat, a mature human T-cell line, Ramos, a human B-cell line, WEHI-231, a murine B-cell lymphoma, and stimulated human peripheral T-cells into progressive stages of the cell division cycle by counterflow centrifugal elutriation. Protocols for using the elutriator to concentrate large volumes of cells prior to separation, the preparation of highly enriched lymphocyte populations at progressive stages through the cell division cycle and conversion parameters from low to high volume rotors are described. Simple dual-staining methods of BrdUrd incorporation and propidium iodide staining for DNA content and subsequent flow cytometry are detailed. Together with [3H]thymidine incorporation data these provide a very accurate determination of cell cycle position of the separated populations.

Mechanism of tracking and cleavage of adduct-damaged DNA substrates by the mammalian 5'- to 3'-exonuclease/endonuclease RAD2 homologue 1 or flap endonuclease 1.

The mammalian 5'- to 3'-exonuclease/endonuclease, called RAD2 homologue 1 or flap endonuclease 1, has a unique cleavage activity, dependent on specific substrate structure. On a primer-template, in which the primer has an unannealed 5'-tail, endonucleolytic cleavage near the annealing point releases the tail intact. Entering at the 5'-end, the nuclease tracks along the entire tail to the point of cleavage. Genetic analyses suggest that this nuclease removes DNA adducts in vivo (Sommers, C. H., Miller, E. J., Dujon, B., Prakash, S., and Prakash, L. (1995) J. Biol. Chem. 270, 4193-4196). Micrococcal nuclease footprinting shows that after tracking the nuclease protects a region of the tail 25 nucleotides long, adjacent to the cleavage site. Substrates with adducts at specific locations were used to assess the mechanism of RAD2 homologue 1 nuclease tracking and its ability to cleave modified DNA. Either a conventional cis-diamminedichloroplatinum (II) (CDDP) or a bulky CDDP derivative was placed within or beyond the region protected by the nuclease. The nuclease cleaved the tail of both substrates. In contrast, a CDDP adduct just adjacent to the expected cleavage point was inhibitory. A CDDP adduct at the very 5'-end of the tail was also cleaved. The nuclease could remove tails containing adducts on the sugar-phosphate backbone. Apparently, the nuclease is designed to slide over various types of damage on single stranded DNA and then cut past the damaged site.

Activation of human monocytes through CD40 induces matrix metalloproteinases.

The activation of monocytes/macrophages to secrete pro-inflammatory cytokines and matrix metalloproteinases (MMPs) is critically important in the development of chronic inflammatory diseases. However, the consequence of interactions between activated T cells and monocytes in these inflammatory processes is not well understood. In this study we have investigated the induction of MMPs in human monocytes by activated T cells. We show that fixed cells and the cell membranes from a T cell line, BMS-2, that expresses high levels of the CD40 ligand gp39 (also called TRAP, TBAM, or CD40L) stimulate both the expression of mRNA and the production of MMPs by human monocytic cells. Activation of monocytes by the human T cells could be significantly inhibited by a F(ab')2 fragment of a neutralizing Ab specific for human gp39, but not by an Ab that recognizes murine gp39. Furthermore, recombinant soluble gp39 (sgp39) alone induced marked increases in the levels of a 92-kDa metalloproteinase (gelatinase) in both the human monocytic cell line, THP-1, and peripheral human monocytes, and induction was blocked by the anti-human gp39 Ab. Pretreatment with IFN-gamma significantly enhanced gp39 induction of MMPs in THP-1 cells but not in peripheral monocytes. Up-regulation of mRNA for the 92-kDa MMP by gp39 could be detected within 6 h of stimulation and was maximal 24 h after treatment. MMP enzymatic activity was detectable in the culture medium 12 to 18 h following stimulation of the cells and remained high through 48 h. These results suggest the interaction of T cells with monocytes/macrophages via the gp39-CD40 counter receptors may be significant in development or maintenance of chronic inflammatory lesions.

Cyclin G1 and cyclin G2 comprise a new family of cyclins with contrasting tissue-specific and cell cycle-regulated expression.

We describe the isolation and characterization of cDNAs encoding full-length human and murine cyclin G1 and a novel human homologue of this cyclin designated cyclin G2. Cyclin G1 is expressed at high levels in skeletal muscle, ovary, and kidney. Following an initial up-regulation from early G1 to G1/S phase, cyclin G1 mRNA is constitutively expressed throughout the cell cycle in T and B cell lines. In contrast, in stimulated peripheral T cells, cyclin G1 mRNA is maximal in early G1 phase and declines in cell cycle progression. Cyclin G1 levels parallel p53 expression in murine B lymphocytes; however, in several human Burkitt's lymphomas, murine lymphocytes treated with transforming growth factor-beta, early murine embryos, and several tissues of p53 null mice, cyclin G1 levels are either inverse of p53 levels or expressed independent of p53. The cyclin G1 homologue, cyclin G2, exhibits 60% nucleotide sequence identity and 53% amino acid sequence identity with cyclin G1, and like cyclin G1, exhibits closest sequence identity to the cyclin A family. Distinct from cyclin G1, the amino acid sequence for cyclin G2 shows a PEST-rich sequence and a potential Shc PTB binding site. Cyclin G2 mRNA is differentially expressed compared to cyclin G1, the highest transcript levels seen in cerebellum, thymus, spleen, prostate, and kidney. In contrast to the constitutive expression of cyclin G1 in lymphocytes, cyclin G2 mRNA appears to oscillate through the cell cycle with peak expression in late S phase.

Loss of normal p53 function confers sensitization to Taxol by increasing G2/M arrest and apoptosis.

The anticancer agent paclitaxel (Taxol) stabilizes tubulin polymerization resulting in arrest in mitosis and apoptotic cell death. Normal human fibroblasts depleted of functional p53 by SV40 T antigen or HPV-16 E6, and primary embryo fibroblasts from p53 null mice showed seven- to ninefold increased cytotoxicity by paclitaxel. Reduced levels of p53 correlated with increased G2/M phase arrest, micronucleation, and p53-independent paclitaxel-induced apoptosis. Surviving cells with intact p53 progressed through mitosis and transiently accumulated in the subsequent G1 phase, coincident with increased p53 and p21cip1,waf1 protein levels. These results are in contrast to studies linking p53 loss with resistance to DNA damaging anticancer agents.

Stimulation of CD40 with purified soluble gp39 induces proinflammatory responses in human monocytes.

CD40 is a glycoprotein of about 50 kDa that plays a crucial role in B cell growth and differentiation. It is found on the surface of B cells, follicular dendritic cells, monocytes, and some endothelial, epithelial, and carcinoma cells. Engagement of CD40 with anti-CD40 mAbs, gp39 expressed on the cell surface or soluble forms of gp39, primes B cells to efficiently respond to subsequent stimulatory signals leading to B cell proliferation, differentiation, and isotype switching. Peripheral monocytes also express CD40 on the cell surface and expression in increased following treatment with IFN-gamma. Using a soluble murine CD8/human gp39 fusion protein (sgp39) we have found that CD40 plays a crucial role in the regulation of monocyte function. Stimulation of human peripheral monocytes with sgp39 induced homotypic aggregation and significantly increased the expression of several cell-surface proteins including CD54, MHC class II, CD86, and CD40. Soluble gp39 also dramatically enhanced monocyte survival, preventing the onset of apoptosis that normally occurs upon withdrawal of serum. Finally, in the absence of any costimulatory molecules, sgp39 stimulated monocytes to produce TNF-alpha, IL-1 beta, IL-6, and IL-8. These results suggest that ligation of CD40 on human monocytes induces phenotypic changes that would be expected to influence T cell activation by the monocyte and also to enhance or prolong inflammatory responses.

Lineage-specific induction of B cell apoptosis and altered signal transduction by the phosphotyrosine phosphatase inhibitor bis(maltolato)oxovanadium(IV).

Protein tyrosine phosphorylation is known to play key roles in lymphocyte signal transduction, and phosphotyrosine phosphatases (PTP) can act as both positive and negative regulators of these lymphocyte signals. We sought to examine the role of PTP further in these processes by characterizing the effects of bis(maltolato)-oxovanadium(IV) (BMLOV), previously known to be a nontoxic insulin mimetic agent in vivo. BMLOV was found to be a potent phosphotyrosine phosphatase inhibitor. BMLOV induced cellular tyrosine phosphorylation in B cells in a pattern similar to that observed following antigen receptor stimulation, whereas little tyrosine phosphorylation was induced in T cells. In B cells, BMLOV treatment resulted in tyrosine phosphorylation of Syk and phospholipase C gamma 2, while sIgM-induced signals were inhibited. By contrast, T cell receptor signals were moderately increased by BMLOV, and the cells displayed greater induction of IL-2 receptor without toxicity. The compound selectively induced apoptosis in B cell lymphoma and myeloid leukemia cell lines, but not in T cell leukemia or colon carcinoma cells. Interleukin-4 plus anti-CD40 antibody treatment of normal human peripheral B cells rescued the cells from BMLOV-induced death. These results suggest that phosphotyrosine phosphatase inhibitors can activate B cell signal pathways in a lineage-specific manner, resulting in desensitization of receptor-mediated signaling and induction of apoptosis.

Activation of p34cdc2 coincident with taxol-induced apoptosis.

Toxicity elicited by the antitumor compound taxol has been linked to irreversible tubulin polymerization, cell cycle block at mitosis, and cell death from apoptosis. We have used pulsed drug exposure of synchronized populations to identify two points, one in transition from G0 to G1 and the other at G2/M of cell cycle, that are most sensitive to taxol-induced cell killing. By analyzing these lesions separately, we have differentiated events related to mitotic block from those that may contribute to apoptosis. The taxol lesion forms rapidly and stably in transition or mitotic cells, because secondary washes to remove residual drug will decrease cytotoxicity except for cells in these populations. Both G2/M cells and G0/G1 transition cells synchronously initiated apoptotic DNA fragmentation within 20 h of pulsed taxol treatment, indicating that a sustained mitotic block is not requisite to initiate cell death. Apoptosis was inhibited by cyclohexamide and by 2-aminopurine and sodium orthovanadate; thus, cell cycle progression appeared requisite for cell death. Taxol treatment of G0/G1 or G2/M cells clearly leads to a block of mitosis followed by a perturbation of tyrosine phosphoprotein regulation; however, protein tyrosine phosphorylation correlated with mitotic block rather than time after drug exposure. Conversely, p34cdc2 kinase activation does not occur at mitotic block but rather 20 h after drug exposure and coincident with DNA fragmentation. Taken together, these results suggest that mitotic block may not be a sufficient signal for taxol-induced apoptosis and that the taxol lesion initiates apoptosis via a phosphoregulation pathway possibly involving the p34cdc2 kinase.

Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle.

The overt effects of the anti-cancer drugs cisplatin (cis-DDP) and taxol appear to be DNA modification and microtubule stabilization respectively, yet the mechanisms by which these drugs elicit tumor cell death are not well understood. In this report cell sensitivities to cis-DDP and taxol were accurately determined as a function of cell proliferation and cell cycle stage. Quiescent fibroblasts restimulated to synchronously enter the cell cycle become maximally sensitive to cis-DDP immediately preceding DNA synthesis, and resistance increases with onset of DNA synthesis. Mid-log proliferating cells were separated into progressive stages of the cell cycle by centrifugal elutriation or by double thymidine (dThd) block. Cells staged by either method are maximally sensitive to cis-DDP in G1, just prior to the onset of DNA synthesis and minimally sensitive in peak DNA synthesis, with entry into S phase resulting in a 2-fold decrease in sensitivity. Cells that remained blocked at the G1/S phase boundary during cis-DDP treatment remain maximally sensitive after release. Sensitivity to taxol increases at 2 points: transiently during transition of normal cells from quiescence to proliferation and steadily as proliferating cells progress from early G1 to late G2. This 3-fold increase in taxol sensitivity through the cell cycle is rapidly reversed upon cell division. Synchronous cells treated with either drug at points of maximum sensitivity initiate apoptotic DNA fragmentation 12-14 hr post-exposure to drug.