Correlations between hyaluronan and epidermal proliferation as studied by [3H]glucosamine and [3H]thymidine incorporations and staining of hyaluronan on mitotic keratinocytes.

The rates of keratinocyte proliferation and synthesis of Hyaluronan (HA) were studied in human whole-skin organ culture by labeling with [6-3H]glucosamine and [3H]thymidine, respectively, to reveal possible correlations between the two functions of the cell. HA distribution in epidermis was examined by staining with a specific probe prepared from cartilage proteoglycan. The keratinocyte proliferation rate was low on the first 2 culture days, but showed a tenfold increase on the third and fourth days while the synthesis of HA proceeded at a relatively stable level throughout the same period. The most intensive staining of HA occurred in the uppermost spinous cell layer, whereas mitotic cells resided in the basal and suprabasal layers. The keratinocytes under various stages of mitosis were surrounded by a HA staining not more intense than that around nondividing basal cells, but a thick pad of HA appeared rapidly between the daughter cells. These findings suggest that newly synthesized HA is associated with the separation of keratinocytes following mitosis but the majority of the synthesis and content of HA in epidermis is involved in other keratinocyte activities such as maintenance of the extracellular space and cell--cell interactions during migration and differentiation.     

LIMITATIONS IN THE USE OF [3H]THYMIDINE INCORPORATION INTO DNA AS AN INDICATOR OF EPIDERMAL KERATINOCYTE PROLIFERATION IN VITRO

The validity of using the incorporation of [³H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [³H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [³H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [¹⁴C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [³H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture

We studied the influence of hydrocortisone (HC) on hyaluronan (HA) metabolism in explants of human skin, a model retaining normal three-dimensional architecture of dermal connective tissue and dynamic growth and stratification of epidermal keratinocytes. The synthesis of hyaluronan and proteoglycans (PGs), and DNA, were determined with ³H-glucosamine and ³H-thymidine labelings, respectively. The total content and histological distribution of hyaluronan was studied utilizing a biotinylated aggrecan-link protein complex. A low concentration of HC (10^?9 M) stimulated the incorporation of ³H-glucosamine into hyaluronan in epidermis by 23% and reduced the disappearance rate of hyaluronan by 25% in chase experiments, resulting in a 74% increase in total hyaluronan (per epidermal dry weight) after a 5-day culture in 10^?9 M HC. On the other hand, a high concentration of HC (10^?5 M) reduced both synthesis (-42%) and degradation (-46%) of epidermal hyaluronan during 24 h labeling and chase periods. The cumulative effect of a 5-day treatment was a 24% decrease of total epidermal hyaluronan. The high dose (10^?5 M) also reduced keratinocyte DNA synthesis and epidermal thickness. In dermis, only the high (10^?5 M) concentration of HC was effective, inhibiting the incorporation of ³H-glucosamine into hyaluronan by 28%. No significant influences on total hyaluronan content or the disappearance rate of hyaluronan in dermal tissue was found. All HC concentrations lacked significant effects on newly synthesized PGs in epidermal and dermal tissues, but reduced the labeled PGs diffusing into culture medium. A low physiological concentration of HC thus maintains active synthesis and high concentration of hyaluronan in epidermal tissue, while high pharmacological doses of HC slows hyaluronan turnover and reduces its content in epidermis, an effect correlated with enhanced terminal differentiation, reduced proliferation rate and reduced number of vital keratinocyte layers. © 1995 Wiley-Liss, Inc.     

Limitations in the use of [3H]thymidine incorporation into DNA as an indicator of epidermal keratinocyte proliferation in vitro

The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation.     

Differential effects of cAMP and cGMP on in vitro epidermal cell growth.

Primary keratinocyte cultures free of dermal fibroblasts were used to investigate the effect of varying cyclic AMP (cAMP) concentrations on epidermal cell function. Addition of 10^?3, 10^?4 or 10^?5 M dibutyryl cAMP to plated cells (day 1) results by day 5 in a dose dependent increase of [³H]TdR incorporation into DNA as determined by increases in both the labeling index and incorporation of ³H label into an isolated DNA fraction. 8-Bromo cAMP, another cAMP analogue, likewise induced keratinocyte proliferation. The proliferative response was dose and time dependent, and 5- to 6-fold increases in ³H label incorporated into DNA were seen at day 6, 8 and up until day 15 of culture. Moreover, elevation of cellular cAMP by addition of cholera toxin, an irreversible stimulator of adenylate cyclase, also demonstrated a time dependent stimulation of [³H]TdR uptake into DNA and increased the labeling index. Specific histochemical staining for keratinaceous protein (Kreyberg technique) demonstrated that elevated cAMP levels also enhance the production of specialized (differentiated) epidermal cells. Determination of the level of cAMP and cyclic GMP (cGMP) by RIA of partially purified fractions of the cultures revealed that addition of 8-bromo cAMP or cholera toxin to the cultures increased the levels of cAMP but not of cGMP. Addition of 8-bromo cGMP to the keratinocytes on day 1 at concentrations of 10^?6, 10^?7 or 10^?8 M had no effect on culture proliferation on days 4, 6 and 8, although qualitative changes in the electron microscopic pattern of the culture stratification and specialization were observed. The results indicate (1) both large and moderate increases in cellular cAMP levels induce keratinocyte culture proliferation and specialization in the absence of fibroblasts or dermal influences, (2) the quantitative enhancement of keratinocyte growth and specialization occurs without apparent participation of cGMP, (3) cGMP may be a qualitative effector of epidermal cell differentiation.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Stratification, specialization, and proliferation of primary keratinocyte cultures. Evidence of a functioning in vitro epidermal cell system

A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.     

Temporal and spatial sequence expression of cytokeratin K19 in cultured human keratinocyte

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.     

Reconstitution of human epidermis in vitro is accompanied by transient activation of basal keratinocyte spreading

Frozen human cadaver skin obtained from the skin bank was thawed and incubated in serum-free medium for 1–2 days, after which the original epidermis could be removed mechanically. Transmission electron microscopic observations showed that the dermal matrix remaining behind contained intact bundles of collagen fibrils but no live cells and that a continuous lamina densa persisted in the basement membrane region. Indirect immunofluorescence analyses demonstrated linear staining of the basement membrane region by antibodies against laminin and type IV collagen and discontinuous staining with antibodies against fibronectin. Scanning electron microscopic observations revealed a normal topographical arrangement of dermal matrix papilla and interspersed crypts on the surface of the matrix. Epidermal cells placed on the dermal matrix attached in 1–2 h and spread by 24 h. After 1 week of culture the epidermis was reconstituted, at which time approximately 30% of the epidermal cells were basal keratinocytes and the remainder were more differentiated keratinocytes. A high degree of differentiation of the reconstituted epidermis was shown by the formation of hemidesmosomes along the basement membrane, the formation of desmosomes characterized by intercellular dense lines, and the presence of a cell layer containing keratohyalin granules. At various times during epidermal reconstitution, cells were harvested and tested in short-term assays for adhesion to fibronectin substrata. During the first several days there was a transient activation of basal keratinocyte spreading analogous to the modulation of keratinocyte spreading that we have observed during epidermal reconstitution in vivo.     

Relative resistance to methothexate by proliferating normal rabbit epidermal cells in vitro

Summary Primary cell cultures of normal rabbit epidermal cells (keratinocytes) were established without the use of enzymatic techniques. Six experiments were carried out on cells from six different rabbits. When these cells were exposed to methotrexate (MTX) for 24 h at 1 μg/ml, proliferation, as measured by cells entering mitosis, was significantly inhibited (P<0.05) in only one experiment. When the dose of MTX was elevated to 100 μg/ml, only two experiments showed significant inhibition of mitosis. This minimal inhibition of mitosis by MTX was contrasted by the dramatic inhibitory effect of this antimetabolite on DNA synthesis. At 1 μg/ml MTX for 24 h, DNA synthesis, as measured by [³H]deoxyuridine uptake, was inhibited >95%. We can conclude that under certain conditions, the rabbit keratinocyte may represent a normal cell type that is inherently resistant to the antiproliferative effects of methotrexate. The research was supported by National Cancer Institute Grant CA 11536.     

Distinct Melanogenic Response of Human Melanocytes in Mono‐culture,in Co‐Culture with Keratinocytes and in Reconstructed Epidermis,to UV Exposure

Striking differences are observed in the melanogenic response of normal human melanocytes to UVA and UVB irradiation depending on culture conditions and the presence of keratinocytes. Exposure of melanocytes co‐cultured with keratinocytes to UVB irradiation triggered, already at low doses (5 mJ/cm²), an increase in melanin synthesis whereas in melanocyte mono‐cultures, UVB doses up to 50 mJ/cm² had no melanogenic effect. Unlike UVB, UVA exposure caused the same melanogenic response in both mono‐ and co‐cultures. Removing certain keratinocyte growth factors from the co‐culture medium abolished the melanogenic response to UVB, but not to UVA exposure. When integrated into the basal layer of a reconstructed human epidermis, human melanocytes similarly reacted to UVA and UVB irradiation as in vivo by increasing their production and transfer of melanin to the neighboring keratinocytes which resulted in a noticeable tanning of the reconstructed epidermis. The presence of a dense stratum corneum, known to scatter and absorb UV light, is responsible for higher minimal UVB and UVA doses required to trigger a melanogenic response in the reconstructed epidermis compared to keratinocyte–melanocyte co‐cultures. Furthermore, an immediate tanning response was observed in the pigmented epidermis following UVA irradiation. From these results we conclude that: (i) keratinocytes play an important role in mediating UVB‐induced pigmentation, (ii) UVA‐induced pigmentation is the result of a rather direct effect on melanocytes and (iii) reconstructed pigmented epidermis is the most appropriate model to study UV‐induced pigmentation in vitro.     

Patterns of mitosis in hamster epidermis.

Alignment of the flattened keratinizing cells of the upper strata of mammalian epidermis leads to the formation of columnar units of structure. In mouse epidermis, mitoses have been found to occur relatively infrequently in the region beneath the center of each cell column where a non-keratinocyte dendritic cell, usually with freatures typical of an epidermal Langerhans cell, is situated. The observed pattern of mitosis could therefore be due either to displacement of central keratinocytes by Langerhans cells or indicate some control of keratinocyte proliferation related either to the Langerhans cells or to the over-lying cell columns. No relationship exists between the position of Langerhans cells and epidermal cell columns in hamster epidermis but measurement of the position of mitosis has shown a reduced frequency of occurrence of mitosis beneath the central region. This pattern of mitosis is therefore unrelated to Langerhans cells and appears to reflect differences in the mitotic potential of basal keratinocytes which could be associated with feedback from the overlying cell columns or with an intrinsic pattern of basal cell activity.     

Calcium-dependent [3H]acetylcholine release and muscarinic autoreceptors in rat cortical synaptosomes during development

A number of presynaptic cholinergic parameters (high affinity [³H]choline uptake, [³H]acetylcholine synthesis, [³H]acetylcholine release, and autoinhibition of [³H]acetylcholine release mediated by muscarinic autoreceptors) were comparatively analyzed in rat brain cortex synaptosomes during postnatal development. These various functions showed a differential time course during development. At 10 days of age the release of [³H]acetylcholine evoked by 15 mM KCl from superfused synaptosomes was Ca²⁺-dependent but insensitive to the inhibitory action of extrasynaptosomal acetylcholine. The muscarinic autoreceptors regulating acetylcholine release were clearly detectable only at 14 days, indicating that their appearance may represent a criterion of synaptic maturation more valuable than the onset of a Ca²⁺-dependent release.     

Biochemical characterization of [3H]norepinephrine uptake in dissociated brain cell cultures from chick embryos

The uptake of [³H]norepinephrine ([³H]NE) was studied in dissociated brain cell cultures prepared from 8-day-old chick embryos using the whole brain (minus optic lobes). Uptake of [³H]NE, 5×10^–9 M, 10 min incubation, in freshly dissociated noncultured embryonic chick brain cells, was detected in 6-day-old embryos; it was temperature and drug (cocaine, metanephrine) sensitive and increased with brain development. In cultured cells, which were assayed at various days in culture, the increase in [³H]NE accumulation per culture was less than that seen in freshly dissociated noncultured embryonic cells. When [³H]NE uptake was expressed per mg protein, a decrease with days in culture was observed, reflecting perhaps a dilution of growth or proliferation of cells not accumulating NE. Metanephrine, 5×10^–6 M, an inhibitor of extraneuronal uptake, inhibited [³H]NE in 5-day-old cultures whereas desmethylimipramine, an inhibitor of neuronal uptake, inhibited [³H]NE uptake in 15- and 20-day-old cultures. Cocaine, another neuronal inhibitor, inhibited [³H]NE at 10 and 15 days only. We interpret these findings to suggest that during early growth in culture most neuroblasts accumulate NE nonspecifically and, as neuronal maturation proceeds, NE accumulation becomes specific.     

Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis

The expression of the large chondroitin sulfate proteoglycan versican was studied in human adult skin. For this purpose, bacterial fusion proteins containing unique portions of the versican core protein were prepared. Polyclonal antibodies against the fusion proteins specifically reacted with versican from a proteoglycan fraction of MG63 osteosarcoma cells. In immunohistochemical experiments, the affinity- purified antibodies localized versican in the stratum basale of the epidermis, as well as in the papillary and reticular layers of the dermis. An apparent codistribution of versican with the various fiber forms of the elastic network of the dermis suggested an association of versican with microfibrils. Both dermal fibroblasts and keratinocytes expressed versican in culture during active cell proliferation. In line with the observation that versican is absent in the suprabasal layers of the epidermis where keratinocytes terminally differentiate, culture conditions promoting keratinocyte differentiation induced a down- regulation of versican synthesis. In Northern blots versican mRNA could be detected in extracts from proliferating keratinocytes and dermal fibroblasts. Comparison of RNA preparations from semi-confluent and confluent fibroblast cultures demonstrated decreasing amounts of versican mRNA at higher cell densities. This inverse correlation of versican expression and cell density was confirmed by indirect immunofluorescence staining of cultured fibroblasts and keratinocytes. The localization of versican in the basal zone of the epidermis as well as the density dependence of versican in cell cultures suggest a general function of versican in cell proliferation processes that may not solely be confined to the skin.     

Efflux of sphingolipids metabolically labeled with [1-3H]sphingosine, L-[3-3H]serine and [9,10-3H]palmitic acid from normal cells in culture

The membrane complex lipids of human fibroblasts and differentiated rat cerebellar granule cells in culture were metabolically radiolabeled with [1-³H]sphingosine, L-[3-³H]serine and [9,10-³H]palmitic acid. A relevant efflux of radioactive sphingolipids and phosphatidylcholine was observed from cells to the culture medium in the presence of fetal calf serum. This event was independent of the concentration and structure of the metabolic precursor administered to cells, and it was linearly time-dependent. The radioactive lipid patterns present in the medium were different from those present in the cells. Radioactive sphingomyelin and ganglioside GM3 containing short acyl chains were the main species present in the medium from human fibroblasts, while sphingomyelin and GD3 ganglioside in that from neuronal cells. In the absence of proteins in the culture medium, the efflux of complex lipids was much lower than in the presence of serum, and the patterns of released molecules were again different from those of cells. This work was supported by COFIN-PRIN, Consiglio Nazionale delle Ricerche (PF Biotechnology), Italy.     

Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3

Keratinocyte growth factor (KGF) activates keratinocyte migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in keratinocytes. Treatment of monolayer cultures of rat epidermal keratinocytes led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within keratinocytes, and a rapid increase of hyaluronan synthase 2 (Has2) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has2 and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has2 and Has3 belong to the targets of KGF in keratinocytes, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of keratinocytes in wound healing, whereas it may delay keratinocyte terminal differentiation.     

Pemphigoid, pemphigus and desmoplakin as antigenic markers of differentiation in normal and tumorigenic mouse keratinocyte lines

Abstract The expression of differentiation stages in a murine epidermal cell transformation model has been investigated as a basis for studies of chemically-induced differentiation. Antibodies in sera of patients with the autoimmune diseases bullous pemphigoid and pemphigus vulgaris exhibit specific reactivity to antigenic determinants of basal and spinous cells, respectively, in sections of mouse and human epidermis. In addition, spinous cells in epidermis are reactive with a mouse monoclonal antibody to desmoplakin, a desmosomal component immunologically distinct from pemphigus. These antibodies were used to identify and attempt to quantify keratinocyte subpopulations in culture based on differentiation stage. Epidermal cell lines were cultured under conditions which favour proliferation (0.02 to 0.04 mm extracellular Ca²⁺, i.e. low Ca²⁺ conditions) or differentiation (0.1 mM to 1.4 mM Ca²⁺), as previously shown using primary cultures of mouse keratinocytes. Two independently-derived normal keratinocyte lines demonstrated Ca²⁺-dependent reactivity with pemphigoid and pemphigus antiserum, like that which has been observed in primary cultures. Furthermore, a Ca²⁺ and time-dependent reactivity with the three antisera was also observed in a papilloma cell line (derived from one of the normal cell lines after treatment in vitro with 7,12-dimethylbenz[α]anthracene). Papilloma cells cultured under conditions of low extracellular Ca²⁺ were comprised of three subpopulations: cells reactive only with pemphigoid anti-serum, cells reactive with pemphigoid and desmoplakin antibody (intracellular location), and cells reactive only with desmoplakin antibody. However, like the normal cell lines, papilloma cells underwent a transition to predominantly a spinous cell population (i.e. reactive with pemphigus and desmoplakin antibody) in response to extracellular Ca²⁺. A slower loss of pemphigoid antibody reactivity was noted in papilloma cells, consistent with an abnormal regulation of differentiation. The attempt to characterize these dynamic transitions from basal to spinous cell subpopulations in culture was considered to be prerequisite for the use of the model to investigate differentiation-inducing agents in carcinoma therapy.     

Role of nuclear proteins on [H]actinomycin D binding during lymphocyte mitogenesis

The uptake of [³H]actinomycin D ([³H]AD) by ConA-stimulated lymphocytes was followed during 96 h of incubation and correlated with the level of nuclear proteins in the nucleus, DNA synthesis and the degree of AD-induced inhibition of RNA and DNA synthesis. During the first 48 h there is a parallel increase of drug binding to cells and a rising level of non-histone proteins (NHP) in the nucleus. During the next 48 h, DNA synthesis occurs, drug uptake decreases and the nuclear level of NHP continues to rise. The level of histones remains constant during 96 h. The variations in cellular [³H]AD uptake during 96 h are not due to changes in cell membrane permeability, since similar variations in drug binding are observed in isolated cell nuclei. NHP, obtained as 0.25 M NaCl extracts of cell nuclei, increase binding of [³H]AD to nuclei isolated from non-stimulated lymphocytes, while histones have no such effect. NHP extracted with phenol, after washing the nuclei with salt and acid solutions, or extracted with 0.25 M NaCl from non-stimulated and stimulated lymphocytes and Chang liver cells are equally active to bind [³H]AD to nuclei of non-stimulated lymphocytes. NHP from Chang cells, purified by DNA-cellulose chromatography using calf thymus DNA, stimulated [³H]AD binding to lymphocyte nuclei, indicating that the drug-binding activity is due to proteins binding to DNA. NHP increase binding of [³H]AD to pure DNA in the absence of histones. The degree of [³H]AD binding to ConA-stimulated lymphocytes during 96 h correlated with the degree of inhibition of RNA and DNA synthesis by AD.     

Reactions of keratinocytes to in vitro millimeter wave exposure.

The effects of millimeter waves (MW) on human keratinocytes were studied in vitro using the HaCaT keratinocyte cell line. MW-induced modulation of keratinocyte function was studied in proliferation, adhesion, chemotaxis, and interleukin-1beta (IL-1beta) production assays. Spontaneous proliferation, adhesion to tissue culture plate, random migration, and IL-8- and RANTES induced chemotaxis were not affected by exposure of cells to millimeter waves under the following conditions: frequency, 61.22 GHz; SAR, 770 W/kg; duration of exposure, 15-30 min. However, MW irradiation resulted in a modest but statistically significant increase in the intracellular level of IL-1beta. These data suggest that exposure of human skin (with keratinocytes being the major component of epidermis) to MW can cause activation of basal keratinocytes resulting in an elevated level of IL-1beta production.