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  • Hydration, volume changes and nuclear magnetic resonance proton

    relaxation times of HeLa S-3 cells in M-phase and the subsequent cell

    cycle

    DENYS N. WHEATLEY1, MARGET S. INGLIS1, MARGARET A. FOSTER2

    and J. EWEN RIMINGTON2

    'Department of Pathology and zBiomedical-l'hysics and Bio-linginecnmi, L'niversity Medical Buildings, Foresteiiiill, Aberdeen . IW 2/1), Scotland, t K

    Summary

    Most mitotic HeLa cells divided into two daughter cells with half the volume of the parent; no additional reduction in volume was detectable during late telophase or the early part of Gx. Synchronous growth throughout the next gener- ation cycle was exponential, 'without an ad- ditional and sudden rise in volume being detect- able as cells entered M-phase. Since the wet weight/dry weight ratio and the protein per unit mass of cells remained constant throughout the cycle, measurements based on volume changes accurately reflected growth parameters and cell water content.

    Nuclear magnetic resonance (n.m.r.) t\ (spin- lattice) proton relaxation times for intracellular water for M-phase cells measured at both 32 MHz (25°C) and 80MHz (37°C) in a Bruker spec- trometer had the same values as normal inter- phase cells. Furthermore, cells arrested in metaphase by nitrous oxide or alkaloids gave the same f, values. Artificial manipulation of cell

    hydration by adjustment of the tonicity of the external medium led to t{ values that correlated well with the level of intracellular water. On this basis, a 40% increase in hydration raised t\ values by a factor of 1-29 at 32 MHz and 1-5 at 80MHz. This is considerably less than the in- crease by a factor of 1-9 in t\ time reported with the 40% rise in cell water accompanying mitosis, measured at 30 MHz and 25 °C under isotonic conditions by others. The protein-synthesizing ability of hydrated cells was reduced to only half the normal level after a doubling of intracellular water.

    The data from these several different analyses, taken together, strongly indicate that the water content of mitotic cells is very similar to that of interphase cells, and that certain features unique to the mitotic phase of the cell cycle cannot be ascribed to an elevated (free) water content.

    Key words: HeLa, n.m.r. spectrometry, hydration, cell cvcle, mitosis, division.

    Introduction

    It has been reported that the t\ nuclear magnetic resonance (n.m.r.) time of mitotic (iW-phase) HeLa cells is increased by as much as a factor of 1 -9 relative to that of interphase cells (Beall el al. 1976). The period of elevation was almost exclusively restricted to M- phase itself, and the inference of a large increase in cell water was confirmed from wet weight/dry weight analysis, which indicated a 40% rise during mitosis. More recently, Ngo et al. (1984), Ngo & Woolley (1986) and Belfi et al. (1986) have also reported

    Journal of Cell Science 88, 13-23 (1987) Printed in Great Britain © The Company of Biologists Limited 1987

    substantially longer (by a factor of 1-9-20X) /, relaxation times for Chinese hamster V79 and mouse C3H10 1/2 cells in .U-phase.

    Changes as dramatic as a 40% rise in cell water content might be considered readily visible by light microscopy, but this is not necessarily the case. In a spherical cell of, say, 16 ^m diameter, a 40% increase in cell volume represents no more than a 9 % increase in diameter. Also the rounding-up of cells going into mitosis and their spreading on return to interphase makes it difficult to appreciate volume changes. How- ever, a sudden and sizeable increase in hydration

    13

  • associated specifically with mitotic division could neatly explain a number of characteristics of mitotic cells, e.g. rounding-up and detachment from the substratum, increased size of amino acid pools (Mac- millan & Wheatley, 1981), increased thermosensitivity (Rao & Engelberg, 1965), and depressed protein syn- thesis (Fan & Penman, 1970). In addition, the notion that increased hydration might be required for success- ful passage of cells through M-phase is attractive because it offers an explanation for the finding that progression can be prevented by the withdrawal of water from mitotic cells in hypertonic media (Stubble- field & Mueller, 1960; Wheatley & Angus, 1973), a procedure that subsequently led to the development of a reversible method of synchronizing cells in division (Wheatley, 1974).

    Our preliminary studies indicated that t\ and tz proton relaxation times increased predictably in HeLa cells progressively swollen in a range of hypotonic media. On this basis, however, a 40 % rise in cell water content increased the t\ time by a factor of only about 1-25-1-30, whereas in studies referred to above a higher factor of 1-9 was found. We have therefore investigated the relationships between cell volume, water content and n.m.r. relaxation parameters more closely for mitotic and non-mitotic (interphase) HeLa cells, and in cells passing synchronously through or arrested at specific stages of the cell cycle.

    Materials and methods

    Cell culturing

    HeLa S-3 cultures were grown in Eagle's Basal medium (BME) in monolayer or a modified suspension version (Mueller et al. 1962). Their generation time averaged 22h. Mitotic collections were made in many experiments by the shake-off method of Robbins & Marcus (1964) without recourse to presynchronization or mitostatic agents. Synchronized populations with >95 % mitotic cells were incubated as suspension cultures for n.m.r. sampling during the subsequent cell cycle. Where mitotic arrest was required, colcemid at l -5xl0~7M was used or, alternatively, exposure to N2O at 5-7 atmospheres for 3 or 6h before shake-off (Rao, 1968) with resuspension in fresh medium to avoid the paramagnetic effects of this gas (Ling, 1983), but normally without the presvnchrony treatments used by Beall's group.

    To synchronize cells in different cycle phases, hydroxyurea at 2XlO~JM or thymidine at 5xlO~3M was added for 16h to suspension cultures before the cells were allowed to enter 5- phase synchronously by resuspension in fresh medium.

    Cell volume measurement

    A Coulter FN' counter coupled to a C-1000 channelyser was used (Coulter Electronics Ltd, Luton, England). For con- firmation, cell diameters were measured at right angles with a carefully calibrated Vickers' image-splitting eyepiece (Vickers' Instruments, York, England).

    Hydration

    Wet/dry weight ratios were obtained from pellets of 2X107

    cells spun out at 3000if for 2 mm and subsequently dried to constant weight over a period of 4 days in a 60-70cC oven.

    Protein

    Protein was estimated by the modified Folin-Ciocalteau method of Oyama & Eagle (1956).

    n.m.r. spectrometry

    This involved a model CXP100 machine (Bruker, Rheinstat- ten, West Germany) with a temperature control unit oper- ating at either 25 or 37(± 0'5)°C. Twenty points were used to measure t\, each point being the mean of four determi- nations. Tau extended to l x / | (1-25). A least-squares fit weighted to remove the skewing effect of the semi-log plot on the errors was used to determine /;. Examination of the residuals showed them to be random and a ̂ goodness-of-fit test showed the data to be monoexponential (P>99-9%). ti was measured by a Carr-Purcell-Meiboom-Gill sequence. Most measurements were made at the more sensitive field strength of 80 MHz, but for comparative reasons 32 MHz was also used. Pellets usually of 1X107 to 2X107 cells were centrifuged at 3000^ for 2 min at 37°C, wicked on the surface after draining and the sides of the n.m.r. tubes were dried with medical-wipe tissue before stoppering. Measurement of / ] , ti and proton density were made within minutes there- after. Procedures other than this simple pelleting of sus- pended cells in normal medium will be described where appropriate in Results.

    Radioactive labelling

    L-[4,5-3H]leucine at 1850 GBq minor ' (50Cimmor ' ) was obtained from Amersham International (Amersham, Bucks, England). The culture medium containing 3X 10~4M-leucine was supplemented with 3 7 K B q m r ' (1 ̂ iCiml"1) of tritiated leucine to achieve labelling of protein, the radioactive leucine not significantly altering total leucine concentration. Measurement of incorporation was made on samples of 5 ml from treated and control cultures taken at 30 and 60min, cooled rapidly in 5 ml ice-cold saline containing 10~zM- leucine. Samples were routinely washed and prepared for scintillation counting and protein estimation as described (see Wheatley, 1982), to give a specific activity of incorporated leucine per fig cell protein.

    Results

    Coulter techniques

    Fig. 1 and Table 1 show volume changes of cells moving through mitosis after a normal shake-off collec- tion at to- Fig. 2 gives the appearances of cells over 60 min after shake-off, with many anaphases having developed by t2o- Most cells were in mid-late telophase by /40 and the vast majority reached early G\ by t^. Physical separation of daughter cells by disruption of their mid-bodies did not occur for approximately a further 2h in many cells, particularly when maintained

    14 D. N. Wieatlw et al.

  • in suspension culture, but they were easily detached by gentle shearing forces, i.e. expressing samples through a pipette five times. The failure of separation in 'unsheared' samples led to the broadening of the M- phase peak with a shift towards the lower channels of

    I 50 100/0

    Channel number

    50 100

    Fig. 1. Coulter channelyser size distribution curves with 'o~'3Mi referring to min of incubation after collection of cells in mitosis (thick lines). The panels on the left to

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