Repeated injection (continuous labelling) experiments in mouse epidermis

J. theor. Biol. (1980) 82,465-472

Repeated Injection (Continuous Labelling) Experiments in Mouse Epidermis CHRISTOPHER S. PO~TEN AND D. MAJOR

Paterson Laboratories and Medical Physics, Christie Hospital and Holt Radium Institute, Withington, ManchesterM20 9BX, England (Received 20 February 1979, and in revisedform 4 September 1979) Theoretical labelling index curves for epidermis have been generated under conditions of repeated tritiated thymidine injection. These curves take into account different injection intervals, circadian fluctuations in labelling and two different models for epidermal proliferation; one based on a homogeneous basal layer with “random” loss initially (later, loss was restricted to late G,), and the other based on a programmed sequential aging of proliferative cellsin a compartmentderived from a minority classof stem cells. These curves have been compared with previously published experimental resultsand with resultsfrom somenew experiments.Both modelsfit the data to someextent provided a meanvalue of T, of about 140 h is assumed.However, the sequentialagingmodelprovidesa slightly better overall fit. A further conclusionis that it is impossibleto makeany accurate statementson the epidermal growth fraction from repeated labelling data.

1. Introduction If tritiated thymidine, [3H]TdR,

is made continuously available to a population of asynchronous cells then the labelling index (LI) rises steadily with increasing time as progressively more cells enter the DNA synthesis (S) phase and become labelled. Continuous labelling data can provide further information on the cell cycle time (TJ and the fraction of cells that is actively involved in cell replacement within the tissue, the growth fraction (GF) (Mendelsohn, 1962), in some cases where exponential growth occurs, and providing T, is relatively short. However, in situations where some cells leave the cycle after mitosis and enter a non-proliferative (non-GF, postmitotic maturing) subpopulation continuous labelling may be much less informative (Steel, 1977). The question relevant there is what model best explains the proliferative behaviour of epidermal basal cells. Continuous availability can be easily achieved in culture but is difficult in vivo where a series of repeated injections of [3HJT’dR spaced a few hours apart is commonly used as an approximation. The alternatives are to administer the 465

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label in the drinking water (where dose is difficult to control) or to use a continuous intravenous infusion (which is technically limiting). The technique becomes difficult and tedious for tissues with long cell cycle times like epidermis. The results are most readily understood if the injection interval used is less than the average duration of the S phase ( Ts) which for a tissue like skin usually means giving 20-40 injections 3-6 h apart. It is likely that the accumulated [3H]TdR dose is toxic to some cells while the stress effects of repeatedly handling the animals and also stretching and holding the skin may stimulate other cells in the basal layer. In either case it seems unlikely that the system remains in an undisturbed steady state during the experiment. We should like to review the published examples of epidermal continuous labelling data together with some new experimental material and to point out that these data can provide little reliable information on T, or GF for epidermis. 2. Description

of the Tissue to be Considered:

Mouse Dorsal Epidermis

The dorsal epidermis of mouse consists of a single layer of proliferative cells (the basal layer) which histologically appears homogeneous but is in fact now believed to be composed of a series of functional groupings of about 10 basal cells (Mackenzie, 1969; Christophers, 1970; Potten, 1974, 1975a, 1976). The average mitotic time in the basal layer (determined in several ways) is about 4-5 days (see review in Potten 1975a where an average value of 116 h is quoted). There are in fact only 5 papers that present epidermal continuous labelling curves (Iversen, Bjerknes & Devik, 1968; Hegazy & Fowler, 1973; Potten, Kovacs & Hamilton, 1974; Denekamp, Stewart & Douglas, 1976; Fukuda ef al., 1978) and these quote T, values in the range 83 h-114 h. The length of S is about 11 h (Potten, 1975a). The basal layer in mouse epidermis contains 13-15% non-keratinocytes (Melanocytes and Langerhans cells, Potten, 1976; Wolff, 1972). Although turnover data for these cells is scarce it seems likely that this turnover is very slow (Mackenzie, 1975). It is not clear whether or not the basal layer contains cells temporarily out of cycle (Go, Lajtha, 1963, 1979, Burns & Tannock, 1971; Fukuda et al., 1978; Hegazy & Fowler, 1973; Potten, 1975a). There is some evidence that migration of cells from the basal layer is not random with respect to the age of cell in the cell cycle; there being an increased probability of migration of later Gi cells (cells more than 60 h into Gi) (Iversen et al., 1968). This is also suggested by the fact that there is a 2-3 day period for maturation of cells on the basal layer before labelled cells appear suprabasally (Potten, 1975b). These observations might indicate that migration is restricted to a

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particular point in G1 or alternatively that there is a 2-3 day maturation in a class of cells that have permanently left the division cycle (post-mitotic maturing cells). Thus the basal layer besides containing cycling keratinocytes (T,, 4-6 days) contains about 15 % more slowly turning over dendritic cells, may contain Go cells that have a slow population turnover, and may also contain post-mitotic maturing cells; these being the oldest are most ready for migration, and are in the non-growth fraction and consequently will be lost from the basal layer early in a continuous labelling experiment. Besides having a non-random migration pattern the basal cells are nonrandomly distributed throughout the cell cycle as evidenced by the marked circadian rhythm (Tvermyr, 1969, 1972). An assumption made in this description of epidermis is that the cells within the cycling keratinocyte population are all of the same type and have the same function i.e. are all stem cells. Experiments where clonal regeneration after irradiation has been studied suggest that only a few (2-7%) of the basal cells are clonogenic stem cells (Potten & Hendry, 1973). Thus many proliferating cells are transit cells with a limited division potential and a limited (short) life span in the basal layer. This indicates that epidermis is similar in its proliferative organisation to bone marrow and testes and similar to that suspected in gastrointestinal mucosa and tongue epithelium (Potten, Schofield & Lajtha, 1979). Inherent in this type of model is the fact that cell migration only occurs from the oldest proliferating class of cells which will outnumber other classes and which will only go through one final S and M phase before maturing and migrating. 3. Published Experimental

Data

The published data (Fig. 1) include experiments on: hairless male and female mice (8 weeks old) using 4 hourly injections of 10 l&i of [3H]TdR

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1. Actual datayoints from the literature for labelling index plotted against time during a series of repeated [ H]TdR injections (3-6 h intervals). The line is fitted by eye. FIG.

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(Iversen et al., 1968) (closed circles on figures); SAS/TO male mice (8-9 weeks old) using 6 hourly injections of 10 FCi [3H]TdR (Hegazy & Fowler, 1973) (triangles); DBA-2 male mice (7-8 weeks old) using 3 hourly injections of 5 p,Ci of [3H]TdR (Potten et al., 1974) (crosses); WHT/Ht male mice (10 weeks old) using 6 hourly injections of 10 &i of [3H]TdR (Denkamp et al., 1976) (squares); dd male mice (11-12 weeks old) using 4 hourly injections of about 50 &i [3H]TdR (Fukuda et al., 1978). These data show: (1) considerable scatter with no well-defined plateau at levels significantly less than 100%; (2) no clear difference between the various strains tested (even hairless); (3) no clear difference between any of the dose or injection regimes except possibly for the very early samples; (4) an initial shallow slope probably due in part to the length of GZ + $4. 4. Kinetic Models Considered

The lines shown in the figures were initially derived by considering the behaviour of cells in 100 separate compartments at one hourly intervals throughout a period approximately equal to a cell cycle time. Later these were derived by appropriate computer programming using 1000 compartments. (A) MODEL

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Figure 2 shows the effect of varying the injection interval on the simplestmodeltested(T,=100h;T,=10h;G~+~M=5h;GF=l.O)with cell loss from the basal layer (into the superficial layers) occurring randomly in relation to the cell cycle giving an exponential age distribution. Once the intervals get longer than T, the slope of the curve is reduced. The initial LI is loo80.

. t;= injection interval (h) r,=lOO(h)

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FIG. 2. Theoretical curves for labelling index (LI%) plotted against time during a series of repeated injections of [“HpdR for different injection intervals (Ti) (3 h, 6 h, 12 h and 24 h). The curves are based on the assumption that the basal layer is homogeneous and that the cells have an average 7’, of 100 h and a T, of 10 h and that they are lost randomly from the basal layer. The growth fraction is assumed to be 1.0.

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less than 10% because of the exponential age distribution of the basal cells. Bearing in mind the points outlined in the section describing the tissue it is not surprising that the theoretical continuous labelling curves generated by this model do not fit the experimental data at all well. If the basal layer were considered to be “contaminated” by cells that were non-replacing and non-ageing then the curves would be expected to plateau at a level equal to the proportion of these non-replacing cells. It is more likely that any “contaminating” minor cell population is in fact being gradually replaced by division, death or migration and this would generate curves that continued to rise with time but at a slower rate. Random loss of cells through the cycle would result in about 45% of the cells being lost in Gi, but as already stated it is unlikely that cell loss occurs with equal probability from all phases. The relatively minor effect of restricting all cell loss to a period between 60 and 80 h into Gi is to shift the theoretical curves slightly to the right. If Gz + $V is lengthened to 15 h then the theoretical curves are shifted slightly to the right and have a longer period at the beginning with a shallow slope. If it is assumed that the rates of entry and exit to S vary sinusoidally with time having a periodicity of 24 h the phases and amplitudes of suitable sine functions can be obtained form the experimental data of Tvermyr (1972). This circadian fluctuation can be accounted for in the theoretical repeated labelling curves. For short injection intervals the circadian rhythms only influence the curves to a minor extent displacing them to the left or right depending on when the first injection is given; the time of maximum labelling (LI max) or minimum labelling (LI min). For longer injection intervals the effect is to alter the slope of the line slightly. Model 1 in its initial form was based on T, of 100 h (taken from the literature) and a random loss of cells with respect to their cell cycle. As can be seen from Fig. 3 (dotted line) the theoretical curve based on this model was a very poor fit to the experimental data (shown in Fig. 3 with some additional results obtained using 6 hourly injections of 5 &i [3H]TdR in hairless (9 weeks old) or DBA-2 (7-8 weeks old) male mice). If the average T, was considered to be 140 h and if migration was restricted to late G1 a curve could be generated which fitted the data slightly better (dashed line, Fig. 3). An alternative curve that provided a similar reasonable fit was one based on a mixture of 60% basal cells with T, of 100 h and 40% with a T, of 200 h. (B)

MODEL

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This model is based on the concept that the epidermis only contains a small fraction (about 10%) of stem cells (Potten & Hendry, 1973) and that

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Loss 50-70 . h Into . G,Tr = 140 h Sequentval ageing 40% post -mbtotlc

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FIG. 3. Data points from the literature with some additional points from experiments on DBA-2 male mice (6 hourly injections) (+ injections begun at 1500 hours, + at 0300 hours), or hairless mice (*), plotted together with the 6 h interval curve from Fig. 2 (random loss) and a 6 h interval curve based on sequential aging with 40% post-mitotic cells. The new data were obtained from autoradiographs of epidermal sheets (Hamilton & Potten, 1972). Also shown (dashed line) is a curve based on the model using a homogeneous basal layer with a r, of 140 h and cell loss restricted to more than 50 h into Gr.

the non-stem cells pass through a sequence of amplifying cell divisions, “aging” as they do so, until they pass into a post-mitotic maturing compartment from which they migrate in a sequence according to their “age” (sequential aging). The model proposes 5 different classes of basal cells; (1) stem cells with long T, values, (2-4) three stages (ages) of amplifying committed cells and (5) post-mitotic cells. The overall T, for the 4 proliferating classes would be in the range loo-140 h. However, any estimate for T, would be influenced by the most rapidly cycling class of cells (probably the later amplifying divisions which outnumber the other classes). Clearly with this type of model the post-mitotic cells cannot be labelled by a single injection of 3HTdR (i.e. are in the non-GF) and being the “oldest” cells are selectively lost first from the basal layer (i.e. during the first few days). Thus, even with significant proportions of non-GF cells continuous labelling curves based on model 2 will eventually all reach 100% labelling. A model based on an overall average T, of 140 h, and a post-mitotic fraction of 0.4 produced theoretical curves that fitted the experimental points well (solid line, Fig. 3). The model used was in fact based on 10% stem cells (Tc = 192 h), 18% amplifying transit cells (T, = 100 h), 31% amplifying transit cells (T, = 144 h) and 41% post-mitotic maturing cells. Results have also been obtained using a daily injection regime (20 FCi [3H]TdR; DBA-2 male mice 7-8 weeks old) with the injections being given at either the time of peak labelling (LI max, 0300 hours) or minimum labelling (LI min, 1500 hours) (Fig. 4). Here two alternative models fitted the data reasonably well (1) model 1 with T, at 140 hours and cell loss restricted to 50-70 hours into

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FIG 4. Data from an experiment where DBA-2 mice were injected once a day at either 0300 hours (time of maximum Iabelling) (crosses) or 1500 hours (time of minimum labelling) (diamonds). Each point represents the mean LI values of at least 12 individual mice (1000 basal cells scored per mouse). The theoretical curves for the sequential aging model (solid lines) and the model assuming a homogeneous basal layer with T, = 140 h and cell loss restricted to 50-70 h into Gr (dashed line) are shown.

G1 and (2) the sequential aging model (model 2) outlined above. Theoretical curves generated by considering a model with 60% of cells with T, of 100 h and 40% with T, of 200 h did not fit the daily injection data as well as the two schemes mentioned above. It is difficult to make any definite statements regarding T, values from repeated labelling curves and virtually impossible to make any statements on GF even when assumptions are made about the proliferative models that apply to epidermis. With the sequential aging model it is possible that the GF could be O-6 and still provide curves which eventually reach 100% labelling. There are four reasons why this technique is unlikely to provide information on growth fraction; (1) the inherent scatter of the data points makes it difficult to define the final slope or plateau; (2) it is unlikely that any non-GF cells are not in fact being gradually replaced (i.e. turning over); (3) curves can be generated based on a programmed sequential aging that will reach 100% labelling even with a significant non-GF; (4) the prolonged nature of the treatment (involving repeated handling, injecting and therefore stressing of the animals) may result in significant disturbance of the steady state kinetics. Cells that are temporarily out of the cycle (for variable periods of time) i.e. in Go would be triggered back into cycle as time passed. Thus it might be expected that all continuous labelling curves for epidermis would eventually reach 100%. Since most T, estimates in skin are dependent on GF and T, may contain the time spent resting out of cycle i.e. Go, or Gi or Ga arrest, it might be difficult to make deductions about T, from continuous labelling in skin particularly since the point where the LI plateaus or reaches 100% is hard to define.

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It is impossible to make firm statements based on continuous labelling data about the proliferative model most applicable to mouse epidermis. However, the data obtained so far would fit a model basedon a programmed sequential aging of cells derived from a minority classof stem cells aswell as, if not slightly better than, a model on a homogeneous cell population with loss restricted to late G1. This work wassupportedby grants from the Medical ResearchCouncil and the Cancer ResearchCampaign.We are grateful to Irene Nicholls, Dorothy Robinson, Joan Bullock and Caroline Chadwick for their help with the new experiments presentedhere. REFERENCES BURNS, F. J. & TANNOCK, I. F. (1971). Cell Tiss. Kinet. 3,321. CHRISTOPHERS, E. (1970). Arch. /din. exp. Derm. 237,717. DENEKAMP, J., STEWART, F. A. & DOUGLAS, B. G. (1976). Cell Tiss. Kinet. 9, 19. FUKUDA, M., OKAMURA, K., FUJITA, S., B~HM, N., ROHRBACH, R. & SANDRITTER, W. (1978). Path. Res.Pract. 163,205. HAMILTON, E. & PO-I-I-EN, C. S. (1972). Cell Tiss. Kinet. 5, 505. HEGAZY, M. A. H. & FOWLER, J. F. (1973). Cell Tiss. Kinet. 6, 17. IVERSEN, 0. H., BJERKNES R. & DEVIK, F. (1968). Cell. Tiss. Kinet. 1, 351. LAJTHA L. G. (1963). J. Cell. camp. Physiol. 62, (suppl.l), 143. LAJTHA, L. G. (1979). Diferenfiation 14,23. MACKENZIE, I. C. (1969). Nature 222,881. MACKENZIE, I. C. (1975). Am. J. Anar. 144, 127. MENDELSOHN M. L. (1962). J. mtn. Cancer Inst.28, 1015. POT-I-EN, C. S. (1971). J. Znuest. Derm. 56,311. PO’I-I-EN, C. S. (1974). Cell Tiss. Kinet. 7,77. Po-~~EN, C. S. (1975~1). J. Invest. Derm. 65,488. PO~EN, C. S. (197%). &it. J. Derm. 93,649. POITEN, C. S. (1976). In Stem Cells of Renewing Cell Populations. (eds. A. B. Cairnie, P. K. Lala & D. G. Osmond,) p. 91. Academic Press. New York PO-I-I-EN, C. S. & HENDRY, J. H. (1973). Inr. J. Radiat. Biol. 24,537. POTTEN, C. S., KOVACS, L. & HAMILTON, E. (1974). Cell Tiss. Kinet. 7, 271. POTTEN, C. S., SCHOFIELD, R. & LAJTHA, L. G. (1979). Biochim. biophys. Actu. 560,281. STEEL, G. G. (1977). Growth Kinetics of Tumors, 351 pp. Oxford: Clarendon Press. TVERMYR, E. M. F. (1969). Virchows Arch. Abt. B. Zellpath. 2, 318. ~‘VERMYR, E. M. F. (1972). Virchows Arch. Abt. B. Zellpath. 11,43. WOLFF, K. (1972). Curr. Prob. Dermat. 4,79.