J. Cell Set. 35, 53-58 (1979) 53Printed in Great Eritain © Company of Biologist! Limited 1979

COMPLEMENTATION OF Gi-PHASE VARIANTS

OF A MAMMALIAN CELL CYCLE

P. M. NAHAPaterson Laboratories, Christie Hospital and Holt Radium Institute,Manchester, M20 gBX, England

SUMMARYComplementation between temperature-sensitive (ts) variants of Balb/c-3T3 defective in

the Gy phase of its cell cycle was measured in the [3H]thymidine-labelling indices of the multi-nucleated cells during incubation at the restricted temperature (38 °C) following cell fusion.One ts variant from each group along the length of the Gx phase was tested for complementation.Varying degrees of complementation were observed between the 4 ts variants tested, judgingby the time of entry into 5-phase and the degree of synchrony attained. At least 3 complement-ation groups were discernible.

INTRODUCTION

Evidence has been accumulating over the past few years to suggest (Prescott, 1976)that some critical regulatory changes (controlling both cell proliferation and dif-ferentiation) take place during the Gx phase of a mammalian cell cycle (Howard &Pelc, 1953; Mueller, 1971). The study of the genetics of the Gx phase is thus a pre-requisite to elucidate the functions controlled in Gv We constructed a tentative 'map'of the G1 phase of Balb/c-3T3 using temperature-sensitive (ts) variant clones (Naha,Meyer & Hewitt, 1975) by measuring their time of entry into S phase (after tem-perature shift-down from 38 to 33 CC). I report here the preliminary results of com-plementation tests performed with one ts variant from each group along the lengthof the Gx phase.

MATERIALS AND METHODS

Cell lines

The cell lines used in these experiments and cultuie conditions of their maintenance werereported before (Naha et al. 1975). The Gx phase ts variants (A83, A123, A92 and A8) wererecloned before complementation tests were performed, the reversion frequencies for platingefficiency being in the order of 1-3 x io"4 at the cell density of 1 x io6/ml.

Cell fusion and complementation

Cell fusion was conducted following the technique described by Harris & Watkins (1965).Equal numbers of cells from each parent (2 x io') grown at 33 °C were fused in the presence of/?-propiolacton-inactivated Sendai virus (HAU 600/ml) and planted in i-ml volumes iniLeighton tubes at a density of 1 x io'/ml and incubated at 38 °C. In a parallel seriesof experiments asynchronous log phase cultures growing at 33 °C were incubated at 38 °Cfor 18 h (roughly one cell generation time). Cells exposed to 38 °C were then fused as above.

54 P- M. Naha

Complementation between the ts variants defective in Gv phase was tested in separate experi-ments : (i) by prelabelling of one parent with [14C]thymidine (14C-TdR, with low specific activity)followed by pulse-labelling with [*H]thymidine (3H-TdR, with high specific activity) of cellsfused and incubated at the restricted temperature of 38 °C; and (ii) by measuring the time ofentry into S phase (by pulse labelling with [3H]thymidine only) of multinucleated cells Qohnson& Harris, 1969) at 38 °C following cell fusion. Pulse labelling with 3H-TdR was performedevery 3 h on replicate cultures; the coverslips were washed, fixed, autoradiographed and stainedwith Giemsa according to the method described before (Naha et al. 1975). Radioactive chemicalswere obtained from the Radiochemical Centre, Amersham.

Since the ts variants were inhibited (Naha et al. 1975) from entering the S phase at 38 °C(the labelling indices of homokaryotic parents after fusion were between 5 and 20 % dependingon the leakiness of the parental cultures) an increase in the percentage of labelled multi-nucleated cells over unlabelled multinucleated cells would indicate (Johnson & Harris, 1969)the extent of complementation and also denote a measure of their time of entry into S phaseand progress through the S phase. It should be emphasized that co-ordination of nuclearlabelling in entering S phase between different cells and synchrony in S are independent andseparate phenomena, the latter (Johnson & Harris, 1969) being ascribed to an indirect effect ofthe fusion method. The percentage of multinucleated cells in a normal population of unfusedwild type 3T3 and its ts variants were counted to be less than o-i, compared to the fused cellswhere the percentage was recorded to be between 58 and 10-2 in different experiments. Onlythose multinucleated cells (3 or 4 nuclei) showing co-ordination of labelling between nucleiwere counted for positive complementarity. Less than 20 % of the multinucleated cells were([3H]thymidine) labelled after fusion within the first 32 h of incubation at 38 °C which waspresumed to be the background indicating no effective complementation during this period, dueprobably to some effect of the fusion (Johnson & Harris, 1969).

In the double-labelling experiment where one parent was pre-labelled with 14C-TdR, theradioactive 14C, because of longer track lengths of the /? particles, resulted in more diffuselabelling over the entire cell contrasting with 3H-TdR labelling which was strictly nuclear.This would render identification of the source of each nucleus in the bi- and multinucleatedcells possible. The strongest indication of complementation between the heterokaryotic Gx-phasevariants was thought to be provided by the unequal radioactive labelling of the nuclei in the multi-nucleated cells after fusion. Johnson & Harris (1969) first reported co-ordination of DNAsynthesis among the nuclei of asynchronous cultures produced after fusion of homo- andheterokaryotic cells in absence of a genetic marker. The fusion products of the Gi-phasevariants used in our experiments were in the strictest term 'homoheterokaryons', since the tsvariants were phenotypically identical with and derived from Balb/c-3T3 cells. Complement-ation between cells from different parents held up at different stages of the Gx phase at 38 CCwould induce each parent to enter S phase at different times and, consequently, show differentdegrees of radioactive labelling during their progress (early or late) through the S phase. Thisis exemplified in Fig. 1. (Relative sequence of the ts variants is shown in the insets of Figs.2 and 3). Ideally, it should be possible to map the Gv phase ts variants by radioactive graincounts in the nuclei of the multinucleated cells as a measure of the distance between thesevariants at the time of their entry into S phase. This, of course, was not the immediate objectiveof the present set of experiments.

RESULTS

The results (Fig. 2) of the first series of experiments showed varying degrees ofcomplementation of A83 with A123, A92 and A8. Judging from the level of 'one-way'complementation observed between A83 and WT (wild-type), A83 and A8 acquirednear optimum level of complementation, though the onset of the S phase was delayedby about 6 h in the latter combination. A generation time of 18 h (measured between-2 S periods) was, however, observed in both cases. These data suggested that thedegree of complementation between A83 and A8 was nearly 100% as compared with

Complementation of Gx-phase variants of a mammalian cell cycle 55

A83 and WT. Since the duration of S phase of wild type 3T3 cells (Naha et al. 1975)was about 9-5 h at 38 °C, only half (around 50%) the population would ideally belabelled at any particular time. The fact that a degree of synchrony (48-2% labelled)was retained in the multinucleated cells of A83 and A8 during the second 5 phase,as compared to A83 and WT, is possibly a reflexion of complementarity between

- * ; *A83+WT ' A83+A8

\

A83+A92

Fig. 1. Result of pulse labelling with 3H-TdR (16 /iCi/ml, 32 Ci/mmol) of cells fusedand incubated at 38 °C. Multinucleated cells ( x 3000) of A83 + A8, A83 + A92 andA83 + A123 are compared with A83 +WT to indicate the unequal radioactive labellingduring S phase.

these parents. It is difficult from these experiments to postulate alternative explanationsfor these bimodal curves, as it is difficult to understand how multinucleated cellstraversed 2 cycles, including mitosis, and appeared in the second 5 phase as multi-nucleated cells. Incubation at the restricted temperature might possibly contributeto this phenomenon when complementary partners were still under the effect ofgrowth restriction.

The percentage of labelled multinucleated cells in A83 and A123, and A83 andA92, showed (Fig. 2) delayed entry into 5 phase, compared with that of A83 andA8. Two rather more significant results observed in these 2 combinations were: (1)

56 P. M. Naha

in the degree of synchrony attained; and (2) in the duration of the S phase. While inthe combination of A83 and A123 the percentage of labelled multinucleated cells inS phase reached 51*8, that in A83 and A92 was 59"2. This is probably another in-dication of close complementarity between these variants. The homokaryons duringthis period did not exceed 20% (data not presented). The labelling index curve in the

60 h

30

E"8

A83 A123 A92 A8

37 49h of incubation at 38°C

61 73

Fig. 2. Labelling indices of multinucleated cells fused and incubated at 38 CC.Complementarity of clone A83+WT (A.. .A) is compared with A83+A8 ( # — # ) ,A83 + A92 ( x • • • x) and A83 + A123 (O——O).

combination of A83 and A123 failed to show 2 distinct peaks; the duration the multi-nucleated cells were in S phase seemed to indicate a longer S phase (12 h). Thiscould happen if the 5 phase of the complementing parents did closely overlap, orelse if perfect complementarity due to close proximity of their genetic functions was ex-tended in time. In the combination of A83 and A92,2 sharp peaks are discernible, the Speriods separated by 15 h. Since the cell generation time was calculated to be about 18 h,the 2 peaks separated by 15 h probably mean a marginal overlapping of the 2 5 phases.

In the second series of experiments where clone A8 was fused with clones A92,A123 and A83, the results (Fig. 3) indicated that the multinucleated cells of A8 andA92, and A8 and A123, entered S phase earlier than those of A8 and A83; this wouldagain, to a certain extent, confirm the relative position of the variants in their functionaltime scale. Consistent with the previous experiment, the multinucleated cells of clonesA83, A123 or A92 in combination with A8 retained (Fig. 3) a degree of synchrony

Complementation of G^-phase variants of a mammalian cell cycle 57

during the second S phase. The labelling indices also showed nearly uniform com-plementarity between clone A8 and the others (between 42-6 and 54-4%). That theS phases (measured during 73 h of incubation) in all 3 combinations were separatedby 18 h was probably an indication suggesting that the clone A8 controls an independentgenetic function unrelated to clones A83, A123 and A92; the same is possibly true of

60

i 30

M

A83 A123 A192 A8

37 49h of incubation at 38 °C

61 73

Fig. 3. Labelling indices of multinucleated cells fused and incubated at 38 °C. Com-plementarity of A8 + A83 ( • — • ) is compared with A8 + A92 (x • • • x) and A8 +A123 (O O).

clone A92 which complemented almost equally with A83 and A8. The extendedS phase in A83 and A123 observed in the previous experiment (Fig. 2) was probablyan indication that they are part of the same genetic function.

The results presented above were from cell fusion experiments where parentalcultures were grown at 33 °C. In the parallel series of experiments where culturesgrowing at 33 °C were preincubated for 18 h at 38 °C before cell fusion, the resultsshowed no qualitative or quantitative difference in the pattern of co-ordinated(unequal) labelling of nuclei in the multinucleated cells compared to those presentedabove. The only difference observed in these experiments were in the lower level oflabelling indices (by nearly a factor of 2) in both the homokaryons and the hetero-karyons. This is attributed to the loss of viability of cells due to incubation at 38 °Cfor 18 h (Naha et al. 1975) prior to fusion. The general similarities in the results of2 parallel series of experiments indicated that the unequal labelling of nuclei in

58 P. M. Naha

the multinucleated cells was not due to the physiological condition of the cells priorto fusion but more probably due to 'genetic' complementarity.

In the double-labelling experiment where one parent was pre-labelled with 14C-TdR before fusion, pulse labelling with 3H-TdR was performed at 24, 36, 48 and60 h after cell fusion. As in the case of single labelling experiments, 3H-labellingcould be detected in less than 5 % of the multinucleated cells up to 36 h after fusion.At 48 h onwards 3H-TdR was observed (around 50%) on all combinations of comple-mentary partners, except the combination of ts A83 and ts A123 where 3H-labellingwas noted to reach 42 percent only at 60 h after fusion. In these experiments also,unequal co-ordinate labelling between all different ts variants was observed.

DISCUSSION

Although the Gx phase of a mammalian cell cycle has not yet been explained byany specific event, there is evidence to suggest that there are some rate controllingsignals during this period which have regulatory function (Johnson & Harris, 1969;Rao & Johnson, 1970). The accepted parameter of initiation of DNA synthesis(S phase) as the only measurement of Gx controls is made doubly ambiguous by thevariability of the G± phase (Prescott, 1976). It has thus become necessary to decipherthe genetic controls of the Gx phase in order to understand the controls in cell proli-feration. Temperature-sensitive variants have provided a powerful tool for the studyof the Gx controls.

The results of the experiments presented above confirm our previous observations(Naha et al. 1975) on the relative positions of the ts variants on a functional time-scalemap. The data also suggest (subject to further verification) that possibly half (up to4 h) of the early Gx phase in Balb/c-3T3 cells is made up of a complex set of functionsforming possibly part of the same gene; the rest perform at least 2 independentgenetic functions as could be detected by the complementation pattern of A92 andA8. We have cloned multinucleated cells (hybrids?) from each of these combinationsgrowing at 38 °C and are presently studying the pattern of segregation in culture, andalso the electrophoretic distribution of their nuclear proteins.

The author acknowledges the technical assistance provided by Mrs K. A. Hewitt. This workwas supported by grants from the Medical Research Council and the Cancer Research Campaign.

REFERENCES

HARRIS, H. & WATKINS, J. F. (1965). Hybrid cells derived from mouse and man: artificialheterokaryons of mammalian cells from different species. Nature, Lond. 205, 640-645.

HOWARD, A. & PELC, S. R. (1953). Synthesis of deoxyribonucleic acid in normal and irradiatedcells and its relation to chromosome breakage. Heredity, suppl. 6, 261-273.

JOHNSON, R. T. & HARRIS, H. (1969). DNA synthesis and mitosis in fused cells. J. Cell Set. 5,603-624.

MUELLER, G. C. (1971). Biochemical perspective of the G1 and S intervals in the replicationcycle of animal cells: a study in the control cell growth. In The Cell Cycle and Cancer (ed.R. Baserga), pp. 269-307. New York: Dekker.

NAHA, P. M., MEYER, A. L. & HEWITT, K. (1975). Mapping of the G t phase of a mammaliancell cycle. Nature, Lond. 258, 49-53.

PRESCOTT, D. M. (1976). The cell cycle and the control of cellular reproduction. Adv. Genet.18, 100-177.

RAO, P. N. & JOHNSON, R. T. (1970). Mammalian cell fusion: studies on the regulation of DNAsynthesis and mitosis. Nature, Lond. 225, 159-164.

(Received 22 May 1978)