Synthesis of DNA in isolated nuclei from differentiated mammalin epidermal cells.

Epidermal spinous and granular cells from the newborn rat neither replicate their nuclear DNA nor proliferate in vitro under conditions which support both processes in basal cells. However, as shown by autoradiography, (3H)thymidine and (14C)bromodeoxyuridine do label nuclei removed from spinous cells but not from granular cells. CsCl density gradient centrifugation of DNA obtained from early differentiated nuclei which had been exposed in vitro to (14C)bromodeoxyuridine, indicated that a considerable level of the tracer was present in the nucleic acid and suggested that replication of the genome had occurred. Therefore, spinous cells appear to retain the capability of reproducing nuclear DNA. Since differentiated cells appear to have the "diploid" level of DNA, these observations point to the replication of DNA as a possible locus of the mitotic inhibition which is coincident with epidermal differentiation.

Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface.

It was previously reported that rat keratinocytes grown at the air-liquid interface on collagen gels or on nylon membranes produce multilayered cultures of uniformly stratified cells, comparable to the epidermis in situ by morphological and biochemical criteria. A protocol has now been developed by which primary human keratinocytes grown for two weeks submerged on microporous nylon membranes and raised to the air-liquid interface for an additional three weeks, exhibit most of the comparable characteristics of the epidermal cells in vivo. Staining with fluorescein isothiocyanate-conjugated monoclonal antibodies indicated the presence of 56,5 and 65-67 kDa keratins as well as filaggrin-type proteins in the upper cellular layers. Desmosomes, lamellar granules and keratohyalin-like granules were observed. Cultures were covered with layers of cornified cells. This study differs from the majority of other investigations on human keratinocytes in that no feeder layers or other biological substrata were used. This system should be useful in toxicological studies of chemicals which are to be applied topically to the skin.

The effect of 2,2'-dichlorodiethyl sulfide on DNA synthesis of a murine stratified keratinocyte culture system.

A primary stratified keratinocyte culture resembling the epidermis in situ was used as a model for studying the effects of exposure to 2,2'-dichlorodiethyl sulfide, or sulfur mustard (SM), on DNA synthesis. A method that distinguishes between semi-conservative (s.c.) DNA synthesis and repair synthesis was used to determine if the former was inhibited following treatment with SM. In this method the density of the newly synthesized DNA was increased by incorporation of 5-bromo-2-deoxyuridine. Density gradient centrifugation was then used to isolate the heavy DNA for quantification. It was demonstrated that topically applied SM in the dose range of 1-10 nmole/cm2 inhibited s.c. DNA synthesis (replication) in a dose and time related manner. Inhibition of DNA replication by SM would result in inhibition of cell division which must be preceded by s.c. DNA synthesis. This failure to replace damaged germinative cells may lead to the destruction of the basal layer which is observed in vivo and in our epidermal culture following exposure to SM. This may also be related to development of vesication observed in exposed intact human skin.

Bis(2-chloroethyl)sulfide (BCES) disturbs the progression of rat keratinocytes through the cell cycle.

Epidermal basal keratinocytes are the primary target in BCES-induced cutaneous injury. DNA synthesis is inhibited by exposure to BCES which could relate to the mustard's cytotoxic effect. The effects of BCES on the cell cycle in keratinocytes synchronized by aphidicolin were investigated. Primary keratinocytes synchronized at the G1/S boundary entered the S, G2, M, and G1 phases at successive times after release from the block. When cells were exposed to 1, 10, or 50 microM BCES in different phases of the cell cycle, cells in the S phase were more sensitive to BCES than cells in the other phases. Keratinocytes exposed to 1 microM BCES at the G1/S boundary exhibited a prolongation of the S phase and a block in the G2 phase. When these cells were exposed to 10 or 50 microM BCES, they did not enter the S phase for up to 12h and the incorporation of thymidine into DNA was inhibited. These results suggest that the blocks in the G2 and G1 phases relate to the cytotoxic effect of BCES on the germinative population of epidermal keratinocytes.

DNA repair in primary human keratinocyte cultures after low level exposure to bis(2-chloroethyl)sulfide.

The literature has reported the appearance and disappearance of single-strand breaks (SSBs) in the DNA of rat keratinocytes after exposure to low levels of bis(2-chloroethyl) sulfide (BCES). Since SSBs are a consequence of depurination or depyrimidination followed by excision of the apurinic or apyrimidinic site and deoxyguanosine (GdR) is the major alkylation site in DNA exposed to BCES, it was hypothesized that repair occurred by a GdR-specific base replacement and not by large section repair. To test this hypothesis, cultures of human keratinocytes (HK) were preincubated with 5-bromo-2'-deoxyuridine (BUdR), a heavy analog of thymidine (TdR) incorporated into replicating DNA, immediately before exposure to BCES. Cultures were incubated postexposure with BUdR, radiolabeled GdR, and/or deoxyadenosine (AdR), to measure base-specific repair, and/or radiolabeled TdR, to measure DNA replication and large section repair. A CsCl density gradient was used to remove any BUdR-containing postexposure DNA replication. Each gradient was assayed for radioactivity (cpm) and DNA content (absorbance at 260 nm). The peak A260 fractions were pooled and rebanded in another CsCl gradient. If DNA repair had occurred, the specific activity (cpm/A260) of the peak A260 fraction in the gradient would be greater than control. After exposure of the cultures to BCES, there was a concentration-dependent increase in the specific activity for [3H]GdR but not [4C]TdR over the concentration range used (20-50 microM BCES). A concentration-dependent increase in specific activity was also detected after [14C]AdR exposure. The literature has also reported that the removal of damaged DNA bases after alkylation is via glycosylases. In this series of experiments, we have demonstrated that cultures of HK exposed to the alkylating agent BCES repair their damaged DNA by the replacement of the damaged base only. In the case of BCES exposure, it is the GdR base and to a lesser extent the AdR base.

Molecular aspects of control in epidermal differentiation.

The mammalian epidermis is organized into layers of structurally different cells--the basal, spinous, granular and cornified layers--which represent steps in the differentiative process that terminates in cornification and desquamation. Investigation of the molecular mechanisms that control this ordered sequence of events provides clues to the etiology of certain epidermal pathologies. DNA synthesis and mitosis are normally restricted to the basal layer. Several substances have been implicated in the mitotic control of epidermal cells, the loss of mitotic activity being the first major step in normal keratinization. Investigations performed in this laboratory indicate that isolated differentiated nuclei can replicate their DNA which they are inhibited from doing in situ. Addition of a high speed supernate from homogenized differentiated cells inhibited this synthetic activity in vitro suggesting the existence of a cytoplasmic inhibitor of DNA synthesis. It is not known whether mitotic inhibition in differentiated epidermal cells is a function of the inhibition of DNA replication. Contrary to previous assumptions, recent experimental evidence clearly indicates that, unlike DNA synthesis, RNA synthesis occurs in differentiated cells. Correlated with this synthetic activity is the observation that a protein rich in histidine is specifically formed in the granular cells. This protein appears to be a component of the keratohyalin granules which fill the cells of the granular layer. Investigations were conducted in this laboratory to determine whether control of the synthesis of this protein occurs at the level of translation or transcription. Translation, in vitro, of mRNA obtained from isolated populations of each epidermal cell type suggested that control of protein synthesis in the differentiating epidermis is transcriptional, i.e. only in the granular cell is there an mRNA for the histidine-rich protein. Transcription, in vitro, of chormatin isolated from the separated cell populations produced RNA with a ratio of cytidine to uracil consistent with the predicted mRNA for this protein thus providing additional support for the hypothesis that epidermal differentiation is controlled at the level of 'gene-readout'.

Cultured keratinocytes in in vitro dermatotoxicological investigation: a review.

The keratinocyte is responsible for the architecture of the epidermis, that portion of the skin that forms the environmental barrier necessary for survival. It also interacts with other cell types in the epidermis in response to various environmental influences. This cell type is used frequently for in vitro cutaneous toxicological investigations as an alternative to whole-animal studies. Several areas of cutaneous research using cultured keratinocytes are germane as regards the scope of this journal. The following areas of biomedical research were reviewed: (1) dermatotoxicology, including environmental chemicals, antiseptics, drugs, metals, and pesticides; (2) immunotoxicology, including inflammation and allergic dermatitis; (3) radiation, including ultraviolet and x-irradiation; and (4) the development of assays as alternatives to whole-animal testing. Due to the abundance of such investigations reported in the last 30 years, this review is limited mainly to reviewing reports published in this decade.

Requirement of hydrocortisone and insulin for extended proliferation and passage of rat keratinocytes.

A procedure for the preparation and cultivation of rat epidermal basal cells from full thickness skin resulted in greater than 99% viability and 90% plating efficiency. However, attempts to subculture monolayers of these epithelial cells grown in medium with serum as the only supplement were totally unsuccessful. When hydrocortisone and insulin were added to the medium, subcultivation of primary growth was obtained. It was demonstrated that hydrocortisone at concentrations as low as 0.1 micrograms/ml was necessary for at least the initial attachment of the cells to the substrate - an essential step in subcultivation. Increasing concentrations of insulin (0.1 to 50 micrograms/ml) caused the rate of proliferation and the cell density to increase, but insulin alone did not support subcultivation.

Failure to observe a relationship between bis-(beta-chloroethyl)sulfide-induced NAD depletion and cytotoxicity in the rat keratinocyte culture.

It has been proposed that the activation of poly(ADP-ribose) polymerase (Papirmeister et al., 1985), which results from the presence of strand breaks in bis-(beta-chloroethyl)sulfide (BCES) damaged DNA, causes depletion in the level of nicotinamide adenine dinucleotide (NAD) leading to cell death. This hypothesis has now been evaluated in the primary submerged culture of rat keratinocytes. The DNA content, the viable cell number, and the proliferative capability (measured by thymidine incorporation) of the culture were all reduced 48 h after exposure to 10 microM BCES. However, the total NAD level, that is, NAD+ plus NADH, was not changed at a dose of BCES lower than 50 microM. This observation was the same in both proliferating and early differentiating cultures. To further test this hypothesis, the modifying effect of inhibiting poly(ADP-ribose) polymerase on cytotoxicity in BCES-exposed cells was investigated. After exposure to 250 microM BCES, the NAD level was reduced to approximately 26 pmol/micrograms DNA. This value was increased to 34-49 pmol/micrograms DNA at both 24 and 48 h postexposure when the cultures were incubated in medium supplemented with 1-10 mM nicotinamide. Nevertheless, the decrease in the DNA content of the culture was not reversed. These results suggest that in the rat keratinocyte culture exposed to BCES, depletion of NAD is not a prerequisite for cell death.

Keratinocytes grown at the air-liquid interface.

A procedure is described which allows primary cultures of rat keratinocytes grown at the liquid-air interface to develop and maintain multilayered strata and to produce highly keratinized sheets morphologically similar to those seen in epidermis in situ. Various substrata were tested and compared as to their ability to support growth and stratification of keratinocytes. It was found that when cultured on plastic surfaces, keratinocytes adhered tightly to the substratum and produced a confluent monolayer that later stratified to two to three layers. Cells plated on Vitrogen 100 collagen failed to reach confluence and, in addition, exhibited the "clustering" phenomenon and deterioration of collagen after 3 to 4 d of growth. Significantly better attachment and spreading were observed for cells grown on rat-tail collagen as compared with plastic and Vitrogen 100 collagen. The best results, including maximal and uniform stratification, were seen in cells grown on a mixture of rat-tail and Vitrogen 100 collagens. The system that was developed in the present study offers a model for use in the study of epidermal toxicity from topically applied environmental chemicals.

Growth and differentiation of primary rat keratinocytes on synthetic membranes.

The attachment, proliferation, and differentiation of primary cultures of keratinocytes isolated from murine epidermis were monitored after purified cell suspensions were seeded and incubated in vitro on various synthetic membranes. Concomitant studies of the effects of attachment factors added to synthetic membranes before use as substrata for keratinocytes were also done. The study demonstrated that a synthetic membrane composed of nylon was superior to other membranes and to plastic control culture vessels in supporting the growth of murine keratinocytes. Although laminin enhanced initial attachment and proliferation of cells on nylon membranes, the untreated substratum was more effective for extended incubation. Stratification and differentiation of these keratinocytes on the nylon substratum was enhanced by raising confluent cultures (7 d) to the air-medium interface so that they were in contact with medium only from the bottom. Cultures raised for 14 d produced many morphologic markers of the epidermis and closely resembled the architecture of this tissue in situ.

Formation of interstrand DNA cross-links by bis-(2-chloroethyl)sulfide (BCES): a possible cytotoxic mechanism in rat keratinocytes.

Interstrand cross-links in the DNA of epidermal basal keratinocytes may be responsible for cell death and consequent vesication in skin exposed to BCES. The formation of cross-links and cytotoxicity were compared when cells in primary monolayer cultures of rat epidermal keratinocytes, synchronized at the G1/S boundary or in the G1 phase of the cell cycle, were exposed to BCES. The dose-responsive formation of cross-links, measured with an ethidium bromide-fluorescence assay, was determined immediately after exposure of cells at either position of the cycle. At 24 hr post-exposure, the level of cross-links in cells exposed at the G1 phase showed had not decreased significantly and was still dose-dependent. However, cells exposed in the G1 phase showed a major decrease in cross-links. Formation of interstrand DNA cross-links appears to be related to the mustard's cytotoxicity.

Macromolecular metabolism of a differentiated rat keratinocyte culture system following exposure to sulfur mustard.

A method for producing a stratified, squamous epithelium in vitro by cultivating rat keratinocytes on nylon membranes has been developed in this laboratory. This epidermal-like culture is being used to obtain a better understanding of the mechanism of skin vesication after topical exposure to the sulfur mustard bis(beta-chloroethyl) sulfide (BCES) dissolved in a selected solvent. Radiolabeled macromolecular precursors (thymidine, uridine, and leucine) have been used to study the effect of BCES on the synthesis of DNA, RNA, and protein, respectively, after topical exposure to the mustard at concentrations of 0.01-500 nmol/cm2 dissolved in 70% dimethyl sulfoxide (DMSO). From these and other studies it has been determined that exposure to even the low concentration of 0.01 nmol BCES/cm2 for 30 min results in significant inhibition of [3H]thymidine incorporation, although complete recovery occurs by 24 h. Significant inhibition of [3H]uridine and [14C]leucine incorporation is observed only after exposure to much higher concentrations of BCES (10-500 nmol/cm2). This suggests a very early lesion in macromolecular metabolism with DNA being the primary target.

Pseudoepidermis, constructed in vitro, for use in toxicological and pharmacological studies.

The purpose of this study was to establish the validity of the stratified, cornified keratinocyte culture as a model for investigating cutaneous toxicities. This pseudoepidermis, grown on a nylon membrane at the air-liquid interface, responded to topical application of a known vesicant similarly to the response of the tissue in vivo. Alterations in the morphology of the in vitro model also resembled pathological changes seen in in vivo models after exposure to this agent. The effects of the skin irritants benzoate and salicylate on protein and DNA synthesis in the culture were also similar to those observed in vivo.

Appearance of interleukin 1 alpha relates DNA interstrand cross-links and cytotoxicity in cultured human keratinocytes exposed to bis-(2-chloroethyl) sulfide.

The utility of an increase in the level of interleukin 1 alpha (IL-1 alpha) as an indicator of cytotoxicity from exposure to bis-(2-chloroethyl) sulfide (BCES) was evaluated in submerged monolayer cultures of human cutaneous keratinocytes. Four-day-old cultures were exposed to 1-100 microM BCES at 37 degrees for 30 min. The amounts of IL-1 alpha in the medium at and in cells 72 h after exposure were measured immunologically with an enzyme-linked immunosorbent assay using monoclonal antibody to human IL-1 alpha. The antibody was conjugated with peroxidase for visualization. Cell viability was measured concomitantly using the trypan blue exclusion technique. The degree of interstrand cross-linking as a measure of damage in the cellular DNA was determined by measuring the fluorescence resulting from the intercalation of ethidium bromide into double-stranded molecules that remained in heat-denatured DNA isolated from cells that had been exposed to BCES. A high correlation was observed between the dose-responsive increase in the level of IL-1 alpha in the medium and in the cells, and the dose-responsive decrease that took place in the fraction of viable cells in exposed cultures. The dose-responsive increase in the interstrand cross-linking found in the DNA of cells immediately after exposure to BCES also correlated with the increase in IL-1 alpha 72 h after exposure. These data suggest that the appearance of IL-1 alpha can be used to quantify the cytotoxicity resulting from BCES-medicated damage to cellular DNA and that degree of cross-linking in the DNA immediately after exposure to BCES is predictive of the level of cytotoxicity in an exposed culture 3 days later.