CELL KINETIC ALTERATIONS IN NORMAL AND NEOPLASTIC CELL POPULATIONS IN VITRO AND IN VIVO FOLLOWING VINCRISTINE*. A REPLY TO DR CAMPLEJOHN'S REVIEW ARTICLE†

It is shown that the lethal action of vincristine (VCR) is dose-dependent and may occur at interphase and mitosis. In general, the VCR dose used to destroy cells must be approximately ten times higher than that used to arrest cells in mitosis at metaphase. There is strong evidence that cells can survive metaphase arrest by a sublethal dose of VCR either completing cytokinesis normally after metabolism of the drug or becoming polyploid because of an impaired mitotic spindle apparatus. These cells are not doomed to die, at least in some cell systems. Furthermore, there is strong evidence in three animal tumour systems (transplantable and autochthonous tumours) that VCR is able to induce in vivo partial synchronization of proliferating tumour cells and/or recruitment of resting cells into the proliferating compartment. Failures to induce partial synchrony in cell populations by VCR may be attributed to resistance to VCR or cytolysis or slow proliferation of cells in badly vascularized tumours. Chemotherapy after synchronization seems to be effective as shown by non-randomized trials in bad-risk patients with solid tumours and acute leukaemias. In a randomized co-operative trial results of the two-drug synchronization protocol in patients with non-Hodgkin's lymphoma of high grade malignancy were statistically better than those of a four-drug protocol (COPP) established empirically. The two-drug protocol was equally effective as the four-drug protocol in Hodgkin's disease. Side-effects were less pronounced with the so-called synchronization scheme.     

AN IN VIVO DOUBLE LABELLING STUDY OF THE SUBSEQUENT FATE OF CELLS ARRESTED IN METAPHASE BY VINCRISTINE IN THE JB-1 MOUSE ASCITES TUMOUR

The fate of cells arrested by Vincristine (VCR) in metaphase is of interest because of the wide use of this substance in cancer chemotherapy and, particularly, in relation to its use in so-called ‘synchronization’ therapy. The present study was designed to answer the question of whether cells blocked in metaphase by VCR subsequently proliferate further or whether they become infertile and die. By means of a double labelling technique with [³H] and [¹⁴C]thymidine (TdR) it was shown that all VCR-arrested metaphases in the JB-1 ascites tumour subsequently became necrotic. These cells did not re-enter a viable G₂ phase following arrest and thus could not take part in a wave of synchronous proliferation. In agreement with earlier studies, VCR was found to lead to arrest in metaphase, not only of cells in or shortly prior to mitosis at the time of VCR administration, but also of the majority of cells which had at this time been in the S and G₂ phase.     

A CRITICAL REVIEW OF THE USE OF VINCRISTINE (VCR) AS A TUMOUR CELL SYNCHRONIZING AGENT IN CANCER THERAPY

Vincristine (VCR) has been used clinically in so-called ‘tumour cell synchronization therapy schedules'. These schedules are based on the assumption that cells, arrested in metaphase by low doses of VCR, subsequently re-enter the proliferative cycle synchronously. However, the evidence that tumour cell synchrony can be achieved under clinical conditions or that ‘cell synchronization therapy schedules’ yield a better therapeutic response than other efficient combination schemes, is scanty. Further, even in experimental systems, the efficacy of VCR as a cell synchronizing agent is disputed. Indeed, in some systems, cells arrested in metaphase by low doses of VCR, do not re-enter a normal proliferative cycle at all following arrest. In addition, the complex nature of the VCR—tumour interaction and the heterogeneous nature of the tumour cell populations against which it is used augurs badly for the successful application of cell synchronization therapy schedules.     

ON THE RELATIONSHIP BETWEEN [3H]CHOLINE UPTAKE ACTIVATION AND [3H]ACETYLCHOLINE RELEASE

The depolarization-induced, calcium-dependent release of [³H]ACh from hippocampal synaptosomes was studied in a superfusion system. Release increased, with increasing depolarization. Barium and strontium effectively substituted for calcium during the depolarization, but magnesium inhibited the release. Releasable [³H]ACh is derived from the sodium-dependent component of the [³H]choline uptake which points out the physiologic importance of sodium-dependent choline transport. It is concluded that [³H]ACh release in this system has the same properties as neurotransmitter release in many other systems. Previous studies have shown that treatments which alter the activity of cholinergic neurons in vivo result in parallel changes in sodium-dependent choline uptake in vitro. When synaptosomes were utilized from animals treated to reduce cholinergic activity, there was a reduced release following the reduced uptake. Conversely, when synaptosomes were taken from animals treated to increase sodium-dependent choline uptake, there was an increase in the release. It is concluded that the changes in sodium-dependent choline uptake in vitro consequent to changes in neuronal activity in vivo result in parallel changes in releasable ACh. A comparison was made between the effect of a number of ions and agents on release and their effect on the in vitro, depolarization-induced activation of sodium-dependent choline uptake. Barium and strontium, ions which substitute for calcium in the release process, support the in vitro activation of uptake. Vinblastine and Bay a 1040, compounds which block release, prevented the in vitro activation of sodium-dependent choline uptake. However, magnesium blocked release in a dose-dependent manner, but did not block the activation of uptake in vitro. Rather, magnesium substituted for calcium and supported the activation of uptake in a dose-dependent fashion. It is concluded that acetylcholine release is not necessary for the activation of choline uptake.     

Genesis and evolution of high-ploidy tumour cells evaluated by means of the proliferation markers p34cdc2, cyclin B1, PCNA and 3[H]-thymidine

Although cell polyploidization is not an infrequent event in mammalian cells and is common in tumours, the mechanisms involved are not well understood. Using the murine B16 cell line as a model, we evaluated the role of some key proteins involved in cell cycle progression: p34^cdc2, cyclin B1 and PCNA. By means of flow cytometry, we showed that both in modal- and in high-ploidy subpopulations, almost all cells were p34^cdc2-positive. In the modal-ploidy subpopulation only 17.1% cells were cyclin B1-positive and 85.6% PCNA-positive; in contrast, in the high-ploidy subpopulation up to 91.8% cells were cyclin B1-positive and 97.3% cells were PCNA-positive (P < 0.001). Immunofluorescence microscopy showed that PCNA was located in the nucleus; p34^cdc2, both in the nucleus and cytoplasm; and cyclin B1 yielded a cytoplasmic spotted pattern with a perinuclear reinforcement. After a 24-h incubation with ³[H]-thymidine followed by withdrawal of the isotope, high-ploidy cells remained labelled 8 days after thymidine withdrawal, in contrast to modalploidy cells. Taken together, our results suggest that polyploid cells are not quiescent, their cell cycle is longer than that of the modal-ploidy population, and they maintain cyclin B1 throughout the cycle, which may contribute to their genesis by impeding the exit from mitosis.     

Griseofulvin interacts with microtubules both in vivo and in vitro

Immunofluorescence microscopy using monospecific tubulin antibody shows that in vivo griseofulvin interferes with the expression of both cytoplasmic and spindle microtubules in tissue culture cells in a concentration-dependent manner. In mouse 3T3 cells cytoplasmic microtubules are destroyed at a griseofulvin concentration of 5 × 10^?5m. At this concentration no increase of the mitotic index is observed but the cells are arrested in interphase, probably due to the destruction of cytoplasmic microtubules. Lowering the drug concentration to 10^?5m allows 3T3 cells to accumulate in c-mitotic (“colchicin-mitotic”) arrest. In HeLa cells the display of spindle microtubules observed in drug-arrested cells appears similar to that seen in normal metaphase cells only at lower griseofulvin concentrations. Higher drug concentrations induce c-mitotic arrest accompanied by an increasing loss of typical metaphase tubulin structures.In vitro polymerization experiments with brain tubulin using both light-scattering and electron microscopy show that in the presence of griseofulvin tubulin can aggregate rapidly in the cold. This behaviour is not found in the absence of the drug. Thus both in vivo and in vitro experiments show that griseofulvin, like other c-mitotic drugs, acts at the level of tubulin polymerization and that its effects are concentration dependent.     

SEPARATION OF HELA CELLS BY COLLOIDAL SILICA DENSITY GRADIENT CENTRIFUGATION : I. Separation and Partial Synchrony of Mitotic Cells

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040–1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.     

AUTORADIOGRAPHIC CONFIRMATION OF THE MITOTIC DIVISION OF EVERY MOUSE JEJUNAL CRYPT CELL LABELLED WITH 3H-THYMIDINE

The question was investigated of whether for crypt epithelia of the jejunum of the mouse all cells labelled after a single injection of ³H-TdR subsequently divide or whether cells exist in the crypt which synthesize metabolic DNA and, therefore, do not undergo division after labelling.
A double labelling experiment was performed with a first injection of ³H-TdR followed 1 hr later by an injection of ¹⁴C-TdR. Then from double emulsion autoradiographs of isolated squashed crypts the number of ³H-only, ¹⁴C-only and double labelled cells and mitoses were counted.
The double labelling produced a narrow, 1 hr wide sub-population of ³H-only labelled cells. This subpopulation of S cells completed its division before labelled cells were lost from the crypts by migration onto the villi. The results showed that this subpopulation of ³H-only cells completely doubled within 3 hr and then remained constant through 6 hr. From this result it was concluded that every cell labelled after a single injection of ³H-TdR divides.
From the same autoradiographs the flow rate through the end of mitosis was measured. From the flow rate and the mitotic index a mitotic duration of 0·5 hr was determined. The agreement of this measured mitotic time with the value calculated from the labelling index, mitotic index and S duration is also strong evidence that every labelled cell divides.
Both experiments show that the intestinal crypt does not contain cells synthesizing metabolic DNA.     

Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle

It is well known that inactivation of Cdk1/Cyclin B is required for cells to exit mitosis. The work reported here tests the hypothesis that Cdk1/Cyclin B inactivation is not only necessary but also sufficient to induce mitotic exit and reestablishment of the interphase state. This hypothesis predicts that inactivation of Cdk1 in metaphase-arrested cells will induce the M to G1-phase transition. It is shown that when mouse FT210 cells (in which Cdk1 is temperature-sensitive) are arrested in metaphase and then shifted to their non-permissive temperature, they rapidly exit mitosis as evidenced by reassembly of interphase nuclei, decondensation of chromosomes, and dephosphorylation of histones H1 and H3. The resulting interphase cells are functionally normal as judged by their ability to progress through another cell cycle. However, they have double the normal number of chromosomes because they previously bypassed anaphase, chromosome segregation, and cytokinesis. These results, taken together with other observations in the literature, strongly suggest that in mammalian cells, inactivation of Cdk1/cyclin B is the trigger for mitotic exit and reestablishment of the interphase state.     

Dynamics of the spatial organization of the chromosome set in cells of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> imaginal disks normally and under the action of the tumor-inducing mutation <Emphasis Type="Italic">Merlin</Emphasis>

Fluorescence of H3-p histone and DAPI was studi ed at different stages of interphase and mitosis in cells of imaginal disks of third-instar Drosophila melanogaster larvae. Three stages differing in the spatial organization of the chromosome set in mitosis were revealed. At the first stage (prophase, prometaphase), the histone 3 phosphorylation level rises, and the volume occupied by the chromosome set in the nucleus increases. The distinctive features of the second stage (metaphase) are a gradual decrease in the histone 3 phosphorylation (the density of phosphorylation remaining constant) and a reduction of the volume occupied by the chromosome set. At the third stage (anaphase, telophase), the intensity and density of the signal from H3-p histone decrease, and the volume occupied by the chromosome set reduces. At this stage, in Mer ⁴ larvae, in contrast to the control strain, the cells prematurely pass from anaphase into telophase. In addition, a subpopulation of cells with an abnormally large volume of nuclear DNA during the G₁ period was revealed in Mer ⁴ larvae. The cells of this subpopulation do not enter into the DNA synthesis and quit the cycle.     

The DNA-based structure of human chromosome 5 in interphase

In contrast to those of metaphase chromosomes, the shape, length, and architecture of human interphase chromosomes are not well understood. This is mainly due to technical problems in the visualization of interphase chromosomes in total and of their substructures. We analyzed the structure of chromosomes in interphase nuclei through use of high-resolution multicolor banding (MCB), which paints the total shape of chromosomes and creates a DNA-mediated, chromosome-region-specific, pseudocolored banding pattern at high resolution. A microdissection-derived human chromosome 5-specific MCB probe mixture was hybridized to human lymphocyte interphase nuclei harvested for routine chromosome analysis, as well as to interphase nuclei from HeLa cells arrested at different phases of the cell cycle. The length of the axis of interphase chromosome 5 was determined, and the shape and MCB pattern were compared with those of metaphase chromosomes. We show that, in lymphocytes, the length of the axis of interphase chromosome 5 is comparable to that of a metaphase chromosome at 600-band resolution. Consequently, the concept of chromosome condensation during mitosis has to be reassessed. In addition, chromosome 5 in interphase is not as straight as metaphase chromosomes, being bent and/or folded. The shape and banding pattern of interphase chromosome 5 of lymphocytes and HeLa cells are similar to those of the corresponding metaphase chromosomes at all stages of the cell cycle. The MCB pattern also allows the detection and characterization of chromosome aberrations. This may be of fundamental importance in establishing chromosome analyses in nondividing cells.     

Potential Pitfalls of [3H]Thymidine Techniques to Measure Cell Proliferation

Pitfalls and artifacts in the use of [³H]thymidine in the measurement of cell proliferation kinetics in vitro and in vivo are reviewed. These pitfalls are of particular significance for the study of inhibitors of cell proliferation including chalones.     

RIBONUCLEIC ACID AND PROTEIN SYNTHESIS IN MITOTIC HELA CELLS

HeLa cells arrested in mitosis were obtained in large numbers, with only very slight interphase cell contamination, by employing the agitation method of Terasima and Tolmach, and Robbins and Marcus. Protein synthesis and RNA synthesis were almost completely suppressed in mitotic cells. Active polyribosomes were nearly absent in mitotic cells as compared with interphase cells treated in the same way. Cell-free protein synthesis and RNA polymerase activity were also greatly depressed in extracts of metaphase cells. The deoxyribonucleoprotein (DNP) of condensed chromosomes from mitotic cells was less efficient as a template for Escherichia coli RNA polymerase than was DNP from interphase cells, although isolated DNA from both sources was equally active as a primer. Despite very poor endogenous amino acid incorporation by extracts of metaphase cells, polyuridylate stimulated phenylalanine incorporation by a larger factor in mitotic cell extracts than it did in interphase cell extracts. These results suggest that RNA synthesis is suppressed in mitotic cells because the condensed chromosomes cannot act as a template, and that protein synthesis is depressed at least in part because messenger RNA becomes unavailable to ribosomes. This conclusion was supported by the demonstration that cells arrested in metaphase supported multiplication of normal yields of poliovirus, thereby showing that the mitotic cell is capable of considerable synthesis of RNA and protein.     

The spatio-temporal dynamics of PKA activity profile during mitosis and its correlation to chromosome segregation

The cyclic adenosine monophosphate dependent kinase protein (PKA) controls a variety of cellular processes including cell cycle regulation. Here, we took advantages of genetically encoded FRET-based biosensors, using an AKAR-derived biosensor to characterize PKA activity during mitosis in living HeLa cells using a single-cell approach. We measured PKA activity changes during mitosis. HeLa cells exhibit a substantial increase during mitosis, which ends with telophase. An AKAREV T>A inactive form of the biosensor and H89 inhibitor were used to ascertain for the specificity of the PKA activity measured. On a spatial point of view, high levels of activity near to chromosomal plate during metaphase and anaphase were detected. By using the PKA inhibitor H89, we assessed the role of PKA in the maintenance of a proper division phenotype. While this treatment in our hands did not impaired cell cycle progression in a drastic manner, inhibition of PKA leads to a dramatic increase in chromososme misalignement on the spindle during metaphase that could result in aneuploidies. Our study emphasizes the insights that can be gained with genetically encoded FRET-based biosensors, which enable to overcome the shortcomings of classical methologies and unveil in vivo PKA spatiotemporal profiles in HeLa cells.     

Inhibition of [3H]Spiroperidol Binding by In Vitro Addition of Ethanol

: The in vitro addition of ethanol at concentrations greater than 0.2% produced an inhibition of [³H]spiroperidol binding. This inhibition was competitive, lowering the affinity without altering the maximum number of sites. Both the rates of association and dissociation were altered such that the ligand spent less time attached to the receptor. Since the binding was competitive, little inhibition was seen at ligand concentrations greater than 300 pm . These results are important for in vitro studies when drugs are dissolved in ethanol, and also for in vivo studies on the effects of ethanol.     

Induction of Blocks in Nuclear Divisions and Overcondensation of Meiotic Chromosomes with Cycloheximide During Conjugation of Tetrahymena thermophila

During conjugation, the micronucleus of Tetrahymena thermophila undergoes five consecutive nuclear divisions: meiosis, third prezygotic division (pregamic mitosis) and two postzygotic mitoses of the synkaryon. The four products of the synkaryon differentiate into macronuclear anlagen and new micronuclei and the old macronucleus is resorbed. The protein synthesis inhibitor cycloheximide, applied during conjugation, induced several developmental blocks. Pairs shifted to the drug during early meiotic prophase (stages I–III) were arrested at prophase. Cycloheximide applied to cells at pachytene (stages IV-VI) to metaphase arrested the conjugants at the stage of modified prometaphase/metaphase with overcondensed, swollen bivalents. In contrast to other systems, in the presence of cycloheximide, separation of chromatids, decondensation of chromosomes and exit from metaphase I were inhibited in both diploid and haploid cells. Pairs shifted to the drug after metaphase I were arrested at postmeiotic interphase after completing one nuclear cycle. The same rule applied to the subsequent cycle; then cells were arrested at the stage of pronuclei, and those pairs with functional pronuclei and synkarya were arrested at the stage of two products of the first postzygotic division (pronuclei were not arrested in nuclear transfer and karyogamy). Only pairs with two products of the first postzygotic division were arrested at the same stage after the cycloheximide treatment. Pairs shifted to cycloheximide during the second postzygotic division were arrested in development of macronuclear anlagen and resorption of old macronuclei. The postmeiotic conjugants pulse-treated with cycloheximide (2 h) yielded heterokaryons retaining parental macronuclei (i.e. they exhibited macronuclear retention).     

The Effect of Dichlorophenyldimethylurea on Light-Stimulated 22Na+, 42K+ and 86Rb+ Absorption in Different Tissues of Phaseolus vulgaris Leaves

The light-stimulated absorption of ⁸⁶Rb⁺ by Phaseolus vulgaris L. leaf slices was found to be sensitive to dichlorophenyldimethylurea in air as well as in nitrogen, whereas light-stimulated ²²Na⁺ absorption in nitrogen was not sensitive to this inhibitor. The absorption of ²²Na⁺ is not affected by light in air. The absorption of ⁴²K⁺ is enhanced by a dichlorophenyldimethylurea-insensitive light effect under anaerobic conditions and further increased by light in the absence of the inhibitor. Light-enhanced ⁴²K⁺ absorption in air was also inhibited by dichlorophenyldimethylurea. Previous work showed that light-stimulated ⁸⁶Rb⁺ and ⁴²K⁺ absorption by Phaseolus vulgaris leaf slices is restricted to the guard cells. The present results are discussed with reference to the effect of light on stomatal opening.     

3H-5-IODO-2'-DEOXYURIDINE TOXICITY PROBLEMS IN CELL PROLIFERATION STUDIES

The toxicity of ³H-5-iodo-2′-deoxyuridine (³H-IUdR) was evaluated by injecting tumor-bearing C3H mice with different concentrations of ethanol (the solvent), different doses of tritium tagged onto either IUdR or thymidine and different chemical doses of IUdR, and then measuring the ³H-IUdR incorporation into duodenal and mammary tumor DNA as well as the cellular kinetics of duodenal crypt cells. Ethanol (37% or less, 0.2 ml/mouse) does not significantly inhibit IUdR incorporation into DNA, and the incorporation after a tritium dose of 75 μCi ³H-IUdR/mouse (about 3 μCi/g body weight) is not less than the incorporation following an injection of 25 μCi ³H-IUdR/mouse when the IUdR dose is below 0.005 μmole per mouse. The toxic effects are primarily due to chemical toxicity from IUdR per se. IUdR, at doses of 0.2 μmoles per mouse does inhibit IUdR incorporation into duodenal and tumor DNA, and the duodenal labeling index and the fraction of labeled mitoses are significantly reduced when 0.013 μmole IUdR per mouse is injected. Also some of the duodenal cells containing IUdR apparently undergo only one post-labeling division and the generation time (T_c) of the cells containing IUdR (25 μCi ³H-IUdR/mouse) is 15.3 hr as compared to 13.3 hr for cells labeled with ³H-T (75 μCi/mouse). This increase in T_c is probably not statistically significant; nevertheless, these results do indicate that one must be exceedingly cautious when using ³H-IUdR as a radiotracer for studies concerned with in vivo cellular kinetics and, at least for C3H mice, the dose should be less than 0.01 μmole per 25 g mouse.     

EFFECT OF CYCLOHEXIMIDE ON THE CELL CYCLE OF THE CRYPTS OF THE SMALL INTESTINE OF THE RAT

A single injection of 1.5 mg/kg of cycloheximide induces a complete disappearance of mitotic activity in rat intestinal crypts within 1.5–2 hr. No significant necrosis of crypt cells is observed even though this phenomenon is accompanied by a marked decrease in uptake of labeled precursors into protein and DNA. Mitoses reappear 6 hr after injection and recovery then follows a cyclic pattern over a period equivalent to one cell cycle, thereby reflecting at least a partial synchronization of cell division. Concurrent use of colchicine, an agent known to induce metaphase arrest, has demonstrated that cycloheximide, while having no apparent effect on cells already in division, prevents the entrance of new cells into visible mitosis. Analysis of the cell cycle suggests that one block initiated by cycloheximide occurs in G₂, presumably as the result of an interference with the formation of protein(s) required for the normal progression of cells from this phase of the cycle into mitosis.     

CELL CYCLE TIME DETERMINATIONS BASED ON LIQUID SCINTILLATION COUNTING OF 3H-TdR LABELED MITOTIC CELLS

Following a 10 min pulse labeling with ³H-TdR, flasks of asynchronous monolayer cultures of Chinese hamster ovary cells were subjected to mitotic selection at 2 hr intervals. The mitotic index of the selected populations was always greater than 90%. Counts per min per cell obtained by liquid scintillation counting were plotted versus time after the pulse label. Comparisons were made between cycle times obtained by the mitotic-scintillation counting method and by the standard per cent labeled mitosis technique. The resulting curves were used for calculations of the cell cycle times and the lengths of G₁, S, G₂ and M phases of the cell cycle. There was less than 2% difference in the cell cycle times obtained using the scintillation method as compared to times calculated from autoradiographic data obtained from individual petri dishes. The mitotic-scintillation counting technique is simple, accurate and rapid and allows the calculation of the cell kinetics parameters within 1 hr of the end of the experiment.