We report the isolation and characterization of a spontaneously immortalized human keratinocyte cell line, NIKS. The cell line is not tumorigenic in athymic nude mice and maintains cell-type-specific requirements for growth in vitro. NIKS cells express steady-state levels of transforming growth factor-alpha, transforming growth factor-beta1, epidermal growth factor receptor, c-myc, and keratin 14 mRNAs comparable with the parental BC-1-Ep keratinocyte strain. BC-1-Ep and NIKS keratinocytes produce similar levels of cornified envelopes and nucleosomal fragmentation in response to loss of substrata attachment. DNA fingerprinting results confirm that the NIKS cells originated from the parental BC-1-Ep keratinocytes. NIKS cells contain 47 chromosomes due to an extra isochromosome of the long arm of chromosome 8, and the near-diploid karyotype appears to be stable with repeated passage. A fully stratified squamous epithelium is formed by the NIKS keratinocytes in organotypic culture. Ultrastructural analysis of both the parental and immortalized keratinocytes reveals abundant desmosomes, hemidesmosomes, and the production of a basal lamina. Our findings with the NIKS cells support the observation that spontaneous immortalization is not linked to alterations in squamous differentiation or the ability to undergo apoptosis. The NIKS human keratinocyte cell line is an important new tool for the study of growth and differentiation in stratified squamous epithelia.

The study of human papillomaviruses (HPVs) in cell culture has been hindered because of the difficulty in recreating the three-dimensional structure of the epithelium on which the virus depends to complete its life cycle. Additionally, the study of genetic mutations in the HPV genome and its effects on the viral life cycle are difficult using the current method of transfecting molecularly cloned HPV genomes into early-passage human foreskin keratinocytes (HFKs) because of the limited life span of these cells. Unless the HPV genome transfected into the early-passage HFK extends the life span of the cell, analysis of stable transfectants becomes difficult. In this study, we have used BC-1-Ep/SL cells, an immortalized human foreskin keratinocyte cell line, to recreate the HPV-16 life cycle. This cell line exhibits many characteristics of the early-passage HFKs including the ability to stratify and terminally differentiate in an organotypic raft culture system. Because of their similarity to early-passage HFKs, these cells were tested for their ability to support the HPV-16 life cycle. The BC-1-Ep/SL cells could stably maintain two HPV genotypes, HPV-16 and HPV-31b, episomally. Additionally, when the BC-1-Ep/SL cell line was stably transfected with HPV-16 and cultured using the organotypic raft culture system (rafts), it sustained the HPV-16 life cycle. Evidence for the productive stage of the HPV-16 life cycle was provided by: DNA in situ hybridization demonstrating HPV-16 DNA amplification in the suprabasal layers of the rafts, immunohistochemical staining for L1 showing the presence of capsid protein in the suprabasal layers of the rafts, and electron microscopy indicating the presence of virus like particles (VLPs) in nuclei from cells in the differentiated layers of the rafts.

Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Significantly, infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer (50). The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. The nonproductive stage of the viral life cycle occurs in the basal compartment of the host epithelium where the virus gains entry, presumably at a site of wounding. In this nonproductive stage, the virus maintains itself as a low-copy-number nuclear plasmid (10). As the host cells differentiate, the productive stage of the viral life cycle occurs in the suprabasal compartment of the epithelium. In the productive stage, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced.The 7,904-bp HPV16 genome contains eight viral genes encoding six nonstructural and two structural proteins. Three of the nonstructural proteins, E5, E6, and E7, are designated as oncoproteins because they are able to transform cells in vitro (22,28,29,33,37,45) and, in the case of E6 and E7, induce tumors in vivo (23,42). Two of the other three nonstructural proteins, E1 and E2, are involved in DNA replication and transcription of the viral genome. E4 is predicted to contribute indirectly to the replication of the viral DNA genome in the productive stage (J. Doorbar, unpublished data; T. Nakahara, personal communication). L1 and L2 are the major and minor capsid proteins, respectively.Unlike E6 and E7, the major viral oncoproteins, the E5 protein of HPV16 is not commonly found in cervical carcinoma cells (3, 4). However, it is considered an oncogene given its ability to transform mouse fibroblasts and keratinocytes, cause the mitogenic stimulation of human keratinocytes, and cooperate with E7 to stimulate proliferation of human keratinocytes (5,28,29,37,45). The E5 gene of HPV16 is an 83-amino-acid hydrophobic membrane protein (8, 21) found localized to the Golgi apparatus, endoplasmic reticulum, and nuclear membrane (11).What led researchers to study the HPV16 E5 protein was that, in contrast to the HPVs, the major transforming protein of bovine papillomavirus type 1 (BPV1) is the E5 protein, a 44-amino-acid highly hydrophobic protein that is localized predominantly to the Golgi and exists as homodimers (13,40,41). The BPV1 E5 protein is able to transform both murine fibroblasts and keratinocytes in transformation assays in vitro (9,

The initial steps of pattern formation in the developing Drosophila eye involve the coordination of cell cycles, changes in cell shape, and the specification of the R8 photoreceptor cell. These events begin several cell rows ahead of the morphogenetic furrow and are positively regulated by secreted signaling proteins and the proneural HLH transcription factor atonal (ato). Two HLH regulatory proteins that function to suppress neuronal development in other tissues, extra macrochaetae (emc) and hairy (h), are expressed ahead of the morphogenetic furrow. While neither h nor emc is required for photoreceptor cell determination, in emc-h-clones the morphogenetic furrow and differentiated eye field advance up to eight ommatidial rows ahead of adjacent wild-type tissue. This indicates that morphogenetic furrow progression and neuronal differentiation are negatively regulated by a combination of anteriorly expressed HLH regulatory proteins.

Hepatocytes from adult rats were maintained in primary culture for up to 10-13 days on nylon meshes coated with a thin layer of rat tail collagen gel. Their ultrastructu're closely resembled that of the liver parenchymal cell in vivo, but hepatocytes in late culture exhibited a pronounced buildup of microfilaments beneath their apical cell surface. Hepatocytes in earl and late cultures secreted albumin, transferrin, and al-acid glycoprotein into the medium; they exhibited a 7-to 10-fold induction of tyrosine aminotransferase activity by dexamethasone; and they expressed an alkaline phosphatase that was similar to that of normal rat liver with respect to its inhibition by the liver enzyme inhibitor L-homoarginine. In addition, the hepatocytes in culture demonstrated phenotypic changes characteristic of fetal liver parenchymal cells. These changes, which paralleled an increase in DNA synthesis, included the expression of and linear increase in the activity of the fetal liver cell enzyme y-glutamyl transpeptidase, an increased production of a1-fetoprotein, and a c ange in the substrate specificity of fructose-bisphosphate aldolase to that of the fetal liver isozyme.It is well-established that, in vivo, hepatocytes from adult rats express fetal liver cell characteristics during hyperplasia and hepatocarcinogenesis (1, 2). Examples include expression by the hepatocyte of the enzyme y-glutamyl transpeptidase (3-5), production of a-fetoprotein (6), and the synthesis of fetal isozymes of enzymes such as fructose-bisphosphate aldolase (7). The functional significance and biochemical regulation of these transitions as they relate to the proliferative state are for the most part unknown and are relatively difficult to study in vivo. We now report a system for maintaining adult rat hepatocytes in primary culture and provide evidence for their rapid transition to a more fetal-like state characterized by the production of fetal proteins and an increase in hepatic DNA synthesis.MATERIALS AND METHODS Reagents. Leibovitz (L-15) tissue culture medium, penicillin/streptomycin mixture, and fetal calf serum were purchased from GIBCO. Hi/Wo/Ba medium (10 times concentrated) was obtained from International Scientific Industries (Cary, IL). Insulin, collagenase (type 1), L-y-glutamyl-p-nitroanilide, glycylglycine, p-nitrophenyl phosphate, D-fructose 1,6-bisphosphate, D-fructose 1-phosphate, and a-glycerophosphate dehydrogenase-triosephosphate isomerase were purchased from Sigma. Dexamethasone phosphate was purchased from Merck, Sharp & Dohme; NADH from Boehringer Mannheim; and L-(+)-homoarginine from Aldrich. y-Glutamyl-4-methoxy-2-naphthylamide was obtained from Vega-Fox Biochemicals (Tucson, AZ), and [methyl-3H1thymidine (40-60 Ci/mmol) was purchased from New England Nuclear.Preparation of Collagen Gel/Nylon Mesh Substratum. Swiss nylon monofilament mesh fabric (Nitex HC3-253) was purchased from TETKO (Elmford, NY). Circular meshes were cut from the fabric to fit the bottoms of 100 X 20 mm tissue culture dishes (Falcon). Mes...