Cytostatic effect of cyclophosphane on esophageal tumors and epithelium of mice

Investigation of cytostatic activity of cyclophosphamide in sarcoma 37 and esophagus epithelium in albino mice with respect to the diurnal rhythm of mitotic activity and the number of labeled nuclei was performed. Apparently the tumour cells in the G1-phase and at the beginning of the S-phase of the mitotic cycle were the most sensitive to the inhibitory effect of this drug. During the completion of the DNA-synthesis period the cell resistance to the action of the cytostatic increased. Cells at the G1-phase of the mitotic cycle were sensitive to the inhibitory action of cyclophosphane in the esophageal epithelium.     

Mode of estrogen action on cell proliferation in CAMA-1 cells: II. Sensitivity of G1 phase population

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate 1) the sensitivity of a specific cell cycle phase or phases to 17 beta-estradiol (E2), 2) the effect of E2 on cell cycle kinetics, and 3) the resultant E2 effect on cell proliferation. In synchronized G1/S cells, E2-induced 3H-thymidine uptake, which indicated a newly formed S population, was observed only when E2 was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E2 added immediately following selection; the total E2-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E2. When E2 was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E2. When E2 addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E2 exposure, the response to E2 was again mixed. When E2 was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E2-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that the early G1 phase and possibly the G2/M phase are sensitive to E2 stimulation, late G1, G1/S, or G2 are refractory; the E2 stimualtion of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, E2 does not facilitate faster cell division; and estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E2 alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E2-modulated G1 events related to tumor growth.     

Synthesis and antitumor properties of carminomycin 13-cyclohexylidenhydrazone

Carminomycin 13-cyclohexylidenhydrazone (CCH) was prepared by interaction of carminomycin 13-hydrazone with cyclohexane. The antiblastomic properties of CCH were studied on mice with transplantable tumors. The preparation was administered intravenously or orally. The studies showed a high antitumor activity of CCH. When CCH was administered intravenously to mice with lymphosarcoma LIO-1, the antitumor effect selectivity of it was practically equal to that of carminomycin. When used in doses equivalent by their toxicity to those of carminomycin, CCH had practically the same inhibitory effect on sarcoma 180 as carminomycin. When used orally in doses equivalent by their toxicity to those of carminomycin, CCH was more effective than carminomycin in treatment of mice with lymphosarcoma LIO-1, sarcoma 180 and lymphadenosis NK/Ly.     

The dependence of the cytokinetic effects of alkylating antitumor preparations on the phase of the hepatocyte cell cycle in regenerating liver]

Effects of alkylating antitumor drugs on resting (G0 phase of cell cycle) and proliferating (G1, S, G2 and M phases) hepatocytes were studied in regenerating mouse liver. Cell cycle kinetics (fraction of labeled mitoses, labeling and mitotic indices) were determined by 3H-thymidine autoradiography. Dipin and fotrin as a DNA-damaging agents attack mainly resting (G0) and proliferating (G1) cells. Effect of the damage results in the inhibition of DNA synthesis and G2 phase arrest in the following mitotic cycle. An alkylating drug phopurin as well as ara-C both suppress the mitotic progression in proliferating hepatocytes and do not influence the resting cells.     

Influence of G2 arrest on the cytotoxicity of DNA topoisomerase inhibitors toward human carcinoma cells with different p53 status

We here report the influence of the cell cycle abrogator UCN-01 on RKO human colon carcinoma cells differing in p53 status following exposure to two DNA damaging agents, the topoisomerase inhibitors etoposide and camptothecin. Cells were treated with the two drugs at the IC90 concentration for 24 h followed by post-incubation in drug-free medium. RKO cells expressing wild-type, functional p53 arrested the cell cycle progression in both the G1 and G2 phases of the cell cycle whereas the RKO/E6 cells, which lack functional p53, only arrested in the G2 phase. Growth-arrested cells did not resume proliferation even after prolonged incubation in drug-free medium (up to 96 h). To evaluate the importance of the cell cycle arrest on cellular survival, a non-toxic dose of UCN-01 (100 nM) was added to the growth-arrested cells. The addition of UCN-01 was accompanied by mitotic entry as revealed by the appearance of condensed chromatin and the MPM-2 phosphoepitope, which is characteristic for mitotic cells. G2 exit and mitotic transit was accompanied by a rapid activation of caspase-3 and apoptotic cell death. The influence of UCN-01 on the long-term cytotoxic effects of the two drugs was also determined. Unexpectedly, abrogation of the G2 arrest had no influence on the overall cytotoxicity of either drug. In contrast, addition of UCN-01 to cisplatin-treated RKO and RKO/E6 cells greatly increased the cytotoxic effects of the alkylating agent. These results strongly suggest that even prolonged cell cycle arrest in the G2 phase of the cell cycle is not necessarily coupled to efficient DNA repair and enhanced cellular survival as generally believed.     

The effect of bleomycin on rapidly proliferating epidermis. A comparative investigation using micro-flow fluorometry, H3Tdr incorporation and a stathmokinetic method (colcemid).

At different time intervals after injection of Bleomycin (BLM) th effect on several kinetic parameters of the hairless mouse epidermis stimulated to proliferate by previous adhesive tape stripping was measured. Micro-flow fluorometry was used to determine the relative number of cells in the various phases of the cell cycle (G1, S and G2). Tritiated thymidine was used to determine labelling indices and grain counts. Colcemid was used to observe the mitotic rate. An initial decrease followed by a subsequent significant increase compared to the non-BLM-treated controls was observed in all parameters studied except the mitotic rate, which remained lower than in the control animals during all 48 hours. The transit time of the cells through the S-phase was initially slightly prolonged, but thereafter it seemed to be shorter than that of the controls. BLM seems to provoke a partial blocking of cells in the G1 phase. When the block is released, a greater number of cells pass through the S phase in partial synchrony at a higher than normal speed. BLM induced a low mitotic rate which remained below the level of that of the normal animals after stripping, even though there obviously was a considerably higher influx of cells from the S phase to the G2 phase. This resulted in a subsequent accumulation of cells in the G2-phase. Thus, BLM has also a blocking effect on the G2-M boundary of the cell cycle. This inhibitory effect of BLM on the mitotic rate was shown to be independent of the effect of BLM on the DNA synthesis. BLM therefore seems to have complex influence on epidermal cell kinetics in vivo. Cells in G1-phase are partially and transiently blocked, but this block is soon released. These cells thereafter pass through the S-phase and pile up in the G2-phase, because BLM also blocks the passage of cells from the G2-phase to mitosis. The overall reduction in cell proliferation seen after BLM in vivo seems mainly to be due to the effect on the G2-M boundray of the cell cycle.     

TIMING OF DNA SYNTHESIS IN THE MITOTIC CYCLE IN VITRO

A study was made of the timing of DNA synthesis in the mitotic cycle under conditions where the average mitotic cycle of populations of human amnion and kitten lung cells in culture was variable. Three types of experiments were performed: (a) Autoradiographs were made of incorporated tritiated thymidine in cells whose mitotic histories were recorded microcinematographically allowing the measurement of telophase + G1 along with the total length of the mitotic cycle. (b) Measurement of the G2 + prophase part of the mitotic cycle was performed under various conditions by exposing cells to tritiated thymidine and observing the increase in labeled metaphases plus anaphases as a function of time. (c) The effect of a change in pH on parts of the mitotic cycle was tested by continuously photographing a single colony of cells first at pH 7.8 and then at pH 7.1. All of our data point to the same conclusion; namely, that within a population of cells with a given generation time, the length of each of the measurable parts of the mitotic cycle has a particular distribution of values and that, when there is a change in the generation time, under our conditions only the T + G1 distribution changes.     

Control of cell progression through the mitotic cycle by carbohydrate provision. I. Regulation of cell division in excised plant tissue

A stationary phase in the root meristem of excised pea roots was established by prolonged carbohydrate deprivation in sterile culture medium. When the stationary phase had been established, cells that had collected in the G1 period of the mitotic cycle were induced to enter the S stage by subjection to relatively short intervals of carbohydrate provision (sucrose spurts). Progression and cycle location of the G1 cells induced to enter S were measured with tritiated thymidine and radioautography. The results indicated that the number of G1 cells induced to enter S increased directly with the spurt duration and that cells could be positioned and retained in the S and/or G2 periods by varying the duration of the spurt. The data support the hypothesis that S and maybe M stages have a relatively larger dependence on carbohydrate availability, and presumably a greater energy requirement, than G1 and G2.     

Cyclin levels during TNF-alpha-induced apoptosis in HIV-infected cells.

Cyclins are cell cycle regulatory proteins. We compared the concurrent kinetics of apoptosis and cyclin expression between HIV-infected cells (J1.1), and uninfected Jurkat cells. Cells were cultured with TNF-alpha and harvested at 24, 48 and 72 hr to examine cyclin expression and DNA content. We found a decline in the levels of the mitotic B cyclin in Jurkat cells (16 to 2%, 48 hr), while in J1.1 cells it was observed in cyclin E (60 to 37%, 72 hr). Because cyclin B is mitotic, results suggest that Jurkat cells undergo apoptosis at G2, while J1.1 cells enter mitosis and then die by apoptosis, as no changes in cyclin B or DNA content at G2M were observed. G1 cyclin E decline in J1.1 cells also suggests that they die after entering mitosis. Based on differences in the cyclins involved, it seems that HIV-1 manipulates the cell cycle to protect J1.1 cells from apoptosis induction at G2, a critical cell cycle phase for HIV replication. Thus, cyclins are useful to characterize points in the cell cycle at which apoptosis is induced, and could become excellent tools to evaluate mechanisms of action of antiretroviral drugs in the cell cycle of HIV-infected cells.     

Cell synchrony techniques. I. A comparison of methods

Selected cell synchrony techniques, as applied to asynchronous populations of Chinese hamster ovary (CHO) cells, have been compared. Aliquots from the same culture of exponentially growing cells were synchronized using mitotic selection, mitotic selection and hydroxyurea block, centrifugal elutriation, or an EPICS V cell sorter. Sorting of cells was achieved after staining cells with Hoechst 33258. After synchronization by the various methods the relative distribution of cells in G1, S, or G2 + M phases of the cell cycle was determined by flow cytometry. Fractions of synchronized cells obtained from each method were replated and allowed to progress through a second cell cycle. Mitotic selection gave rise to relatively pure and unperturbed early G1 phase cells. While cell synchrony rapidly dispersed with time, cells progressed through the cell cycle in 12 hr. Sorting with the EPICS V on the modal G1 peak yielded a relatively pure but heterogeneous G1 population (i.e. early to late G1). Again, synchrony dispersed with time, but cell-cycle progression required 14 hr. With centrifugal elutriation, several different cell populations synchronized throughout the cell cycle could be rapidly obtained with a purity comparable to mitotic selection and cell sorting. It was concluded that, either alone or in combination with blocking agents such as hydroxyurea, elutriation and mitotic selection were both excellent methods for synchronizing CHO cells. Cell sorting exhibited limitations in sample size and time required for synchronizing CHO cells. Its major advantage would be its ability to isolate cell populations unique with respect to selected cellular parameters.     

Role of inducible heat shock protein 70 in radiation-induced cell death

We previously demonstrated the protective effect of inducible heat shock protein 70 (Hsp70) against gamma radiation. Herein, we extend our studies on the possible role of Hsp70 to ionizing radiation-induced cell cycle regulation. The growth rate of inducible hsp70-transfected cells was 2-3 hours slower than that of control cells. Flow cytometric analysis of cells at G1 phase synchronized by serum starvation also showed the growth delay in the Hsp70-overexpressing cells. In addition, reduced cyclin D1 and Cdc2 levels and increased dephosphorylated phosphoretinoblastoma (pRb) were observed in inducible hsp70-transfected cells, which were probably responsible for the reduction of cell growth. To find out if inducible Hsp70-mediated growth delay affected radiation-induced cell cycle regulation, flow cytometric and molecular analyses of cell cycle regulatory proteins and their kinase were performed. The radiation-induced G2/M arrest was found to be inhibited by Hsp70 overexpression and reduced p21Waf induction and its kinase activity by radiation in the Hsp70-transfected cells. In addition, radiation-induced cyclin A or B1 expressions together with their kinase activities were also inhibited by inducible Hsp70, which represented reduced mitotic cell death. Indeed, hsp70 transfectants showed less induction of radiation-induced apoptosis. When treated with nocodazole, radiation-induced mitotic arrest was inhibited by inducible Hsp70. These results strongly suggested that inducible Hsp70 modified growth delay (increased G1 phase) and reduced G2/M phase arrest, subsequently resulting in inhibition of radiation-induced cell death.     

Cell cycle-specific glucocorticoid growth regulation of a human cell line (NHIK 3025)

It has been reported that the human cell line NHIK 3025 has a specific cytoplasmic glucocorticoid receptor. When these cells were exposed to glucocorticoids, the cell cycle time was prolonged. Cells, synchronized by mitotic selection, were subjected to the synthetic glucocorticoid dexamethasone throughout the cell cycle. Only cells exposed in the first half of G1 phase had a lengthened cell cycle time. Most of the prolongation was also located within the G1 phase. The dexamethasone growth inhibition was reversible and could be detected only in the cell cycle where the cells were exposed to the steroid. DNA-histograms of asynchronous cells were recorded by flowcytometry at various times after steroid exposure. These histograms also showed G1 phase sensitivity and G1 phase prolongation after exposure to dexamethasone. Our results thus indicate that these cells have a dexamethasone-sensitive restriction point in mid-G1 phase of the cell cycle.     

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.     

Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.     

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.     

Effect of rubomycin and carminomycin on suppressor cells participating in the regulation of the immune response

The effect of rubomycin and carminomycin on induction of suppressor cells in immunization of mice with high doses (2 . 10(9) cells) of sheep red blood cells (SRBC) or with the allogeneic spleen cells was studied. When the spleen cells of syngeneic animals hyperimmunized with SRBC were administered to intact cells, a marked specific suppression of both the antibody formation and the reaction of the delayed type hypersensitivity were observed. A decrease in the suppressor activity or its complete elimination as a result of exposure of the spleen cell donors to the antibiotics was indicative of a relatively selective effect of these drugs on the suppressor cells. This effect of the antibiotics increased after treatment of the spleen immune cells with the anti-T-serum and complement. However, the blocking effect of the antibiotics on the suppressor cells was not strictly specific, since they were detected in the syngeneic and allogenic systems with the cell transfer.     

The centriolar cycle in polykaryons containing prematurely condensed chromosomes or telophase-like nuclei]

In fused interphase-mitotic cells, either interphase nuclei are induced to premature chromosome condensation (PCC) or mitotic chromosomes are induced to telophase-like nuclei (TLN) formation. This study concerns structural and functional changes in centrioles of fused cells in which PCC or TLN are induced. Embryonic pig kidney cells were fused using a modified PEG-DMSO-serum method. Cell cycle period of the nuclei was determined before cell fusion using double-labeling autoradiography. Polykaryons containing desirable type of PCC or interphase nuclear combination in TLN were selected on the basis of isotope labeling after being embedded in epon. Selected cells were cut into serial sections and studied under electron microscope. The data obtained showed that centrioles at every interphase period undergo mitotic activation when their nuclei are induced to PCC. They acquire fibrillar halo and form half-spindles. Daughter centrioles at G1, S and G2 periods are also capable of mitotic activation when separated from their mother centriole. Inert centrioles were found in some cells with G1-PCC. When mitotic nuclei are induced to TLN formation, their centrioles also become inactivated. They lose fibrillar halo and mitotic spindles break down. Some mitotic centrioles develop features characteristic of interphase period such as satellites and vacuoles. Induced nuclear and centriolar changes are simultaneous and may be controlled by the same factor. Mitotic factor of mitotic cell partner which induces PCC may also induce interphase centrioles to mitotic activation. Degradation of the mitotic factor leading to TLN formation may also cause the loss of the mitotic activity of centrioles and disorganization of mitotic spindles.     

The role of protein metabolism in glucocorticoid-lnduced prolongation of G1 phase in human NHIK 3025 cells

It has previously been found that human NHIK 3025 cells have a glucocortiocoid-sensitive restriction point in mid-G1 phase of the cell cycle. When these cells were synchronized by mitotic selection and exposed to dexamethasone before the restriction point, G1 phase was prolonged whereas the rest of the cell cycle was unperturbed by the hormone. These observations were confirmed by flowcytometric mesurements of synchronized cells in the present study. Cells that received dexamethasone (10^?6 M) just after mitotic selection had a 4 hour prolongation of both G1 and the total cell cycle. However, the general rates of both protein synthesis and protein degradation were found not to be altered by the hormone, i.e., the rate of protein accumulation in dexamethasone exposed cells was equal to that of control cells. Dexamethasone exposed NHIK 3025 cells were found to be larger than control cells at the time of cell division. This is a direct consequence of a prolonged cell cycle duration with no change in general protein metabolism. It thus appears that the dexamethasone-induced prolongation of G1 phase is the result of a steroid-regulated G1 specific process(es) leading toward DNA replication, a process that does not alter general protein accumulation.     

Different apoptotic responses and patterns in adhering and floating neoplastic cell cultures: effects of microtubule antagonists

The relationship between apoptotic progression and cell cycle perturbation induced by microtubule-destabilising (vinblastine, Colcemid) and -stabilising (taxol) drugs was studied in two mesenchyme-derived neoplastic cell lines, growing as suspension (Jurkat) and monolayer (SGS/3A) culture, by morphocytochemical and biochemical approaches. The same kind of drug induced different effects on the cell kinetics (proliferation, polyploidisation, death) of the two cell lines. In floating cells, the drugs appeared more effective during the S phase, while in adherent cells they were more effective during the G2/M phase. Moreover two distinct neoplasia-associated apoptotic phenotypes emerged: the first pattern was the typical one and was found in cells with a low transition through the S/G2 phase (Jurkat), and the second one was mainly characterised by a cell death derived from micronucleated and mitotic cells, as a consequence of a low transition through the M/G1 phase (SGS/3A). Our data show that the machinery required for the trigger and progression of apoptosis is present in every cell cycle phase, also in conditions of karyological alterations (aneugenic micronucleations). On the other hand, a different sensitivity of the two microtubular components (interphasic network and mitotic spindle) appears to be related to the anchorage-dependence or -independence during the cell growth disturbances after exposure to antimicrotubular drugs.     

Paclitaxel sensitization of multidrug-resistant cells to chemotherapy is independent of the cell cycle

BACKGROUND: Recent studies have shown that paclitaxel (Taxol) is an active chemotherapeutic in the treatment of small cell lung cancer. Paclitaxel binds to tubulin and prevents depolymerization. This causes cells to arrest in the G(2)/M phase of the cell cycle, resulting in sensitization of cells to drug or radiation treatment. METHODS: A drug-resistant H69 small cell lung cancer subline was established. Cytotoxicity of cisplatin and chlorambucil was determined using the MTT cell viability assay and distribution of DNA in the cell cycle. DNA distribution was analyzed by flow cytometry after treatment with paclitaxel or the other tubulin-binding drugs, vinblastine and navelbine. RESULTS: The H69-EPR drug-resistant subline was resistant to epirubicin (sixfold) and was cross-resistant to cisplatin (7.5-fold) and chlorambucil (7.5-fold). Pretreatment with paclitaxel or vinblastine, but not navelbine, sensitized the subline to cisplatin and chlorambucil (P < 0.05), with no effect on parental H69 cells. Sensitization was dose dependent and occurred at doses below those that caused a G(2)/M block in the cell cycle. CONCLUSION: Sensitization of drug-resistant cells by paclitaxel was not associated with its ability to cause a G(2)/M block in the cell cycle. Sensitization by paclitaxel and vinblastine, but not navelbine, which preferentially targets mitotic tubulin, suggests that sensitization may involve changes in the tubulin-dependent intracellular transport processes rather than changes in mitotic tubulin and the G(2)/M block.