Role of proliferation and apoptosis in net growth rates of human breast cancer cells (MCF-7) treated with oestradiol and/or tamoxifen.

Studies on growth regulation in vitro to a large extent rely on comparison of growth curves. However, these do not discriminate between the relative contributions of the mitotic rate and the apoptotic rate to the net growth rate. In the present study, differential effects of 17beta-oestradiol (E2, 10(-8) M) and/or tamoxifen (TAM, 10(-6) M) on proliferation and apoptosis have been examined and related to growth curves of a subline of the human breast cancer cell line MCF-7 adapted to grow at low serum concentrations. Counting of cells and scoring of labelling and apoptotic indices were performed at the start of the experiment and 3, 6 and 9 days after changing the experimental media. The results demonstrate that apoptosis in this subline is constitutively expressed, that E2 protects (at least partly) against apoptosis and stimulates proliferation, resulting in an increased (net) growth rate, and final cell pool size, and that TAM has a weak cytostatic effect and stimulates apoptosis strongly, resulting in a decreased (net) growth rate and final cell pool size. When E2 and TAM are added simultaneously to the medium, the cytotoxic effect of TAM is partly counterbalanced by the protective role of E2, resulting in a reduced apoptotic rate that, however, is at a higher level than in cultures grown with E2 only. As the cytostatic role of TAM is partly abolished by E2, the combined effect of E2 and TAM results in a final (net) growth rate and cell pool size intermediary to cells grown with E2 or TAM alone.

Tissue kinetics, ion transport, and recruitment of mitochondria-rich cells in the skin of the toad (Bufo bufo) in response to exposure to distilled water.

Mitochondria-rich cells (MRC) of the amphibian epidermis are responsible for active chloride uptake at low external salinity, and new MRCs are recruited in response to exposure to distilled (deionized) water. The time-course of this recruitment, the tissue kinetics and ion transport have been studied in toads (Bufo bufo) immediately before, and after 2,7, and 14 days exposure to distilled water. General epidermal structure was not affected. However, the numbers of MRCs per mm2 (DMRC) increased throughout the experiment as revealed by staining of epidermal sheets with AgNO3 (Ag) or methylene blue (MB). Part of the increased DMRC was accounted for by an increase in MRC subpopulation(s) that stained neither with Ag nor MB. The cell birth rate (Kb) decreased and cell loss by moulting (Kd) increased without any significant change in epidermal cell pool size, indicating a reduced apoptotic rate. The increase in DMRC was accompanied by a 3-fold increase in Cl- current (ICl). At day-2 there was a transient reduction in the ICl per MRC. H+ secretion was progressively reduced during prolonged exposure to distilled water. Thus, at day-2 MRCs appeared incompletely differentiated as indicated by decreased ICl and H+ flux per MRC, and by the increased proportion of MRCs unstained by Ag or MB. Full Cl- (but not H+) transport capacity, was restored at day-7. We conclude that increased DMRC following exposure to low external Cl-, rather than being due to an increased Kb, is the combined effect of a decreased apoptotic rate and an increased rate of differentiation, where 'morphological differentiation' precedes 'functional differentiation'.

Ultrastructure and neuron-specific enolase (NSE) immunohistochemistry of Merkel cells in normal toad epidermis, and following ablation of the pars distalis of the pituitary gland.

The present study was performed in order to re-examine the possible Merkel cell dependency upon the anterior pituitary by a combined ultrastructural and NSE immunohistochemical analysis of Merkel cells in normal toads, and following pars distalis ablation. Ultrastructurally, toad Merkel cells appeared similar to those in previous reports. They were found in normal as well as in operated toads, but with lower frequency in the latter group. By NSE immunohistochemistry, Merkel cells were seen in normal toads only. Even in individual, operated toads, in which Merkel cells were found with relative high frequency by electron microscopy, no NSE could be demonstrated immunohistochemically. It is discussed whether the amount of NSE present depends upon the physiological state of the Merkel cell and in some cells occurs in so low an amount that the NSE antigen cannot be detected by the immunocytochemical method applied. If it is so, the failure to demonstrate Merkel cells in the operated toads by means of the specific marker NSE may be interpreted as an inhibition of NSE expression following pars distalis ablation. This interpretation combined with the lower number of Merkel cells found ultrastructurally in operated toads support a previous indication of an influence of the anterior pituitary--directly or indirectly--upon toad Merkel cell function.

Epidermal tissue homeostasis: apoptosis and cell emigration as mechanisms of controlled cell deletion in the epidermis of the toad, Bufo bufo.

In normal, non-expanding toad epidermis more cells are produced than needed to replace cells lost by moulting. By implication, cell deletion additional to moulting must take place. This paper deals with the mechanisms by which the "surplus" of cells is deleted, taking advantage of the fact that the ratio between cell birth rate (Kb) and the rate of desquamation (Kd), which in normal toads is 2 to 3, can be manipulated. In toads deprived of the pars distalis of the pituitary gland it is decreased to 0.2 to 0.3, and in toads with hydrocortisone pellets implanted into the subcutaneous lymph space it is increased to 7 to 10. Thus, structures candidates for the morphological manifestation of the deletion process should occur rarely in toads in which the pars distalis has been removed and frequently in toads with hydrocortisone pellets implanted. Categorization and enumeration of such structures by light microscopy in the epidermis from operated, normal, and hormone-treated toads were performed. The incidence of structures referred to as "dark cells" and "omega-figures" were found to correlate relatively well with the Kb/Kd-ratio. A subsequent ultrastructural analysis - on a cell-by-cell basis - of "dark cells" showed these to reflect various stages of apoptosis. The duration of the apoptotic process was calculated to be approximately 7 h. Light- and electron microscopy of "omega-figures" combined with histochemical observations of PSA-lectin binding were interpreted as reflecting a release of cells from the basal epidermis and their final elimination within the dermis. It is concluded (i) that apoptosis is an important mechanism of controlled cell deletion, (ii) that emigration to, and elimination in, the dermis is a possible deletion mechanism, and (iii) that necrosis is unlikely to play a role in controlled cell deletion.

Epidermal tissue homeostasis. III. Effect of hydrocortisone on cell pool size, cell birth rate and cell loss in normal toads and in toads deprived of the pars distalis of the pituitary gland.

It has previously been shown that in toad epidermis the cell birth rate (Kb) exceeds the rate of cell loss through moulting (Kd) and that the 'surplus' of cells seems to be removed in a controlled manner. Assuming that the epidermis is non-expanding, a Kb/Kd ratio greater than 1 indicates that cell deletion additional to desquamation takes place. In normal toads this ratio is 2-3. Following implantation of hydrocortisone pellets into intact toads (release rate, 18 micrograms/g toad/d), the Kb/Kd ratio, over a period of 14 d of hormone treatment, had increased to about 7, due mainly to an increased Kb and to a lesser extent to a decreased Kd. No change in the epidermal cell pool size had taken place. It was previously shown that, following removal of the pars distalis of the pituitary gland, the Kb/Kd ratio decreased with time, due to a decreasing Kb and an increasing Kd, eventually leading to a decreased epidermal cell pool size. In this paper it is shown that, in pars distalisectomized toads with hydrocortisone pellets implanted, the Kb/Kd ratio is restored to control levels by a restoration of the Kb as well as the Kd. The results differ from those of previous studies in which ACTH or adrenocorticosteroids were administered discontinuously (by injection). Thus, by experimental manipulation, different Kb/Kd ratios can be obtained: low (less than 1, pars distalis ablation), medium (2-3, normal toads) and high (7, hydrocortisone implantation). The potentiality of this unique situation in analysing the important question of how the 'surplus' cells are deleted is discussed.

Epidermal tissue homeostasis. I. Cell pool size, cell birth rate and cell loss by moulting in the intact toad, Bufo bufo.

Toad epidermis is a suitable model for studies on tissue homeostasis because cell pool size, influx into and efflux from the cell pool can be easily determined. The cell pool size was obtained by cell counting on photomicrographs, the influx (cell birth rate) was assessed by the metaphase-arrest technique, and the efflux (cell loss by moulting) assessed by counting the number of cells in the corneal layer and recording of intermoult periods. The importance of the methods for assessing these parameters per square unit of skin surface is emphasized. These parameters were studied in eight groups of ten adult male toads sacrificed at various hours of the day. There were minor variations in the cell birth rate, fluctuating around a mean of 26 cells/mm2/hr (obtained at the metaphase collection period from 11.00-14.00 hours). By summation of the cell productions during the eight metaphase collection periods of 3 hr, and extrapolation to an intermoult period (time between two moults), a calculated cell production of about 6340 cells/mm2 in 10.3 days was obtained, whereas the cell loss at each moult was only 2370 cells/mm2. Thus the cell production rate exceeds the rate of cell loss through moults by a factor of 2.7 Arguments are presented that the 'surplus' of cells produced cannot be permanently accommodated within the living epidermis. Consequently a cell deletion rate beyond that by moulting of about 4000 cells/mm2 in 10.3 days or 16 cells/mm2/hr can be calculated. These results are discussed in relation to current concepts of tissue homeostatic mechanism(s). The results are consistent with the hypothesis that controlled cell deletion may be a tissue homeostatic mechanism complementary to controlled cell divisions.

Epidermal tissue homeostasis. II. Cell pool size, cell birth rate and cell loss in toads deprived of the pars distalis of the pituitary gland.

Following removal of the pars distalis of the pituitary gland in toads, epidermal efflux from the stratum corneum recruitment cell pool (i.e. production of corneal layers) is greatly increased. In this investigation the cell birth rate is studied by means of the metaphase arrest technique, as a function of time after pars distalis ablation. The method allows assessment of the total cell production over 14 days after the operation, to be compared with the total efflux and changes in the epidermal cell pool size. Whereas in intact toads the rate of cell production exceeds that of cell loss by moulting by a factor of 2.7, the 'surplus' of cells neither being used for formation of corneal layers nor permanently accommodated within the living epidermis, a 'balance sheet' of efflux and influx indicates that following pars distalis ablation all cells produced are also used for the (excessive) formation of corneal cell layers. The observations lend further support to the hypothesis that controlled cell deletion is a tissue homeostatic mechanism complementary to controlled cell divisions.

Structure of the toad epidermis during the moulting cycle. II. Electron microscopic observations on Bufo bufo (L.).

Changes in the ultrastructure of the toad epidermis during the moulting cycle are described on the basis of 17 skin preparations fixed in consecutive phases of the cycle. Our previous light microscopical findings that morphological changes are mainly restricted to a short period prior to and after shedding are confirmed. Differentiation of zonulae occludentes in the new replacement layer after shedding is described and discussed in relation to the changes in ion permeability after the moult. Changes in appearance and distribution of filaments and of two different types of granules during the moulting cycle are described and discussed in relation to current views on amphibian keratinization; it is concluded that the initial phase of keratinization in the toad is very rapid and with a high degree of synchrony, whereas the laying-down of interfibrillar, central dense matrix in the new stratum corneum takes up to 24 hours and is less synchronous. The separation of the old stratum corneum from the replacement layer is gradual; it may be accomplished by rupture of "pillars" bearing the desmosomal complexes between stratum corneum and the replacement layer, or by breaking within the desmosomes themselves. Observed changes in granular content of the replacement layer are considered of no importance for this process, since the time sequence of discharge into the subcorneal space is not correlated with the initiation of separation. Other possible mechanisms of separation are discussed.