[1] Isolation and utilization of epidermal keratinocytes for oncogene research

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KERATINOCYTES

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Isolation and Utilization of Epidermal for Oncogene Research

3

Keratinocytes

B y ANDRZEJ A . DLUGOSZ, A D A M B . GLlCK, TAMAR TENNENBAUM, WENDY C. WEINBERG, a n d STUART H . YUSPA

Introduction Keratinocytes have been widely used as target cells for testing the activity of oncogenes in epithelial neoplasia. Many experimental studies have utilized cultured mouse skin keratinocytes, where in vitro results can be analyzed in the context of a substantial experience in carcinogen-induced mouse skin tumors.a More recent experiments have employed keratinocytes derived from human skin, oral cavity, or cervix, where results can be directly extrapolated to cancers or warts originating in the corresponding epithelia. 2-6 Several laboratories have utilized hamster or rat keratinocytes 7-m in analyses of oncogenes. The contribution of various oncogenes and tumor suppressor genes to the development of epithelial neoplasia has been reviewed, u In experimental epidermal carcinogenesis, oncogenic ras recombinant constructs have received considerable attention because this gene family is frequently mutated in mouse and human skin cancers. Studies of mouse keratinocytes have also revealed the biological consequences of expressing v-los and derivatives, neu, mutant p53, and transforming growth factor c~ (TGF-c0. These studies have focused on the participation of oncogenes as mediators of premalignant progression and malignant conversion. The effects of oncoS. H. Yuspa and A. A. Dlugosz, in "Physiology Biochemistry and Molecular Biology of the Skin" (L. A. Goldsmith, ed.), p. 1365. Oxford University Press, New York, 1991. 2 S. P. Banks-Schlegel and P. M. Howley, J. Cell Biol. 96, 330 (1983). J. S. Rhim, T. Kawakami, J. Pierce, K. Sanford, and P. Arnstein, Leukemia 2, 151S (1988). 4 C. D. Woodworth, S. Waggoner, W. Barnes, M. H. Stoler, and J. A. DiPaolo, Cancer Res. 50, 3709 (1990). S. L. Li, M. S. Kim, H. M. Cherrick, J. Doniger, and N. H. Park, Carcinogenesis (London) 13, 1981 (1992). 6 B. J. Aneskievich and L. B. Taichman, J. Invest. Dermatol. 91, 309 (1988). 7 G. T. Diamandopoulos and M. F. Dalton-Tucker, Am. J. Pathol. 56, 59 (1969). R. D. Storer, R. B. Stein, J. F. Sina, J. G. DeLuca, H. L. Allen, and M. O. Bradley, Cancer Res. 46, 1458 (1986). 9 S. A. Bayley, A. J. Stones, and C. G. Smith, Exp. Cell Res. 177, 232 (1988). l0 K. Yamanishi, F. M. Liew, Y. Hosokawa, S. Kishimoto, and H. Yasuno, Arch. Dermatol. Res. 282, 330 (1990). i1 S. H. Yuspa, Cancer Res. 54, 1178 (1994).

METHODS 1N ENZYMOLOGY,VOL.254

Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.

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genic D N A viruses have also been tested in keratinocytes, and particular emphasis has been directed toward the expression of simian virus 40 (SV40) and specific subtypes of human or bovine papilloma viruses (HPV, BPV) or their transforming sequences. Other studies have tested the biological activity of herpes, adeno-, and Epstein-Barr viruses (EBV) in cultured human keratinocytes. 6'12-15 Because skin keratinocytes are easily isolated and culture conditions are well defined, many oncogene studies have been performed on primary or low passage cells, a quality making keratinocyte studies unique and highly valid with regard to in v i v o biology. Several nonneoplastic m o u s e 16'17 and human as'19 keratinocyte continuous cell lines have been developed. Although these cell lines have extended the keratinocyte model to laboratories less familiar with primary culture techniques, they should not be considered normal target cells since chromosomal and genetic aberrations have been detected in the parental lines. Benign neoplastic mouse 2°,21 and immortalized human 3,22keratinocyte cell lines have also been used in oncogene studies, particularly for the analysis of premalignant progression. While details of the techniques employed in studies testing the influence of oncogenes on keratinocytes are outlined below, several general conclusions have become evident from the results. Oncogenes which appear to function early in the transformation process (e.g., ras, HPV E6 and E7, SV40 T antigen) alter the terminal differentiation program of keratinocytes, allowing transformed cells to grow in an environment which triggers terminal differentiation of normal c e l l s . 2'23-26 Oncogenes which function later in 12 A. Razzaque, O. Williams, J. Wang, and J. S. Rhim, Virology 195, 113 (1993). 13 C. W. Dawson, A. B. Rickinson, and L. S. Young, Nature (London) 344, 777 (1990). 14 R. Fahraeus, L. Rymo, J. S. Rhim, and G. Klein, Nature (London) 345, 447 (1990). 15 j. S. Rhim, G. Jay, P. Arnstein, F. M. Price, K. K. Sanford, and S. A. Aaronson, Science 227, 1250 (1985). 16 S. H. Yuspa, B. Koehler, M. Kulesz-Martin, and H. Hennings, J. Invest. Dermatol. 76, 144 (1981). 17 B. E. Weissman and S. A. Aaronson, Cell 32, 599 (1983). ~8M. L. Goldaber, J. Kubilus, S. B. Phillips, C. Henkle, L. Atkins, and H. P. Baden, In Vitro Cell Dev. Biol. 26, 7 (1990). 19 p. Boukamp, R. T. Petrussevska, D. Breitkreutz, J. Hornung, A. Markham, and N. E. Fusenig, J. Cell Biol. 106, 761 (1988). 20 j. R. Harper, D. R. Roop, and S. H. Yuspa, Mol. Cell. Biol. 6, 3144 (1986). 21 G. P. Dotto, J. O'Connell, G. Patskan, C. Conti, A. Ariza, and T. J. Slaga, Mol. Carcinog. 1, 171 (1988). 2z p. Boukamp, E. J. Stanbridge, D. Y. Foo, P. A. Cerutti, and N. E. Fusenig, Cancer Res. 50, 2840 (1990). 23 S. H. Yuspa, A. E. Kilkenny, J. Stanley, and U. Lichti, Nature (London) 314, 459 (1985). 24 j. B. Hudson, M. A. Bedell, D. J. McCance, and L. A. Laiminis, J. Virol. 64, 519 (1990). 25 M. S. Barbosa and R. Schlegel, Oncogene 4, 1529 (1989). 26 R. Schlegel, W. C. Phelps, Y. L. Zhang, and M. Barbosa, EMBO J. 7, 3181 (1988).

[1]

KERATINOCYTES

5 Epidermis ~ ~ D e r m i s 4°C 0.25% Trypsin

Wash Newborn Mice Amputate Limbs Longitudinal Incision

Fold Back Skin with Forceps

Stretch Skin to Square Shape

Float Skin for 15-24 Hours

. _

Mince Epidermis Filter Through Nylon Gauze

Stir in Complete Medium for 30 Minutes at 37 °

Peel Back Dermis from Epidermis with Forceps

FIG. 1. Protocol for isolating primary keratinocytes from newborn mouse skin. See texl for additional details.

neoplastic progression (e.g., los, mutant p53, ras) profoundly influence keratinocyte growth control. 2°-22'27 Culture Techniques

Murine Keratinocytes Newborn mouse epidermis yields a large number of cells (5-10 × 10~'/ epidermis), with a 30-40% plating efficiency. Mice are sacrificed by CO2 narcosis 1-4 days postpartum (prior to the appearance of hair), soaked in Betadine for 5 min, rinsed twice in 70% ethanol (v/v), and kept on ice. Using an aseptic technique, limbs and tails are amputated, a longitudinal incision is made from tail to snout, and skin is peeled off the carcass using forceps (Fig. 1). Skins are stretched out with the dermis facing down in the bottom of 150-mm culture dishes, and stored at 4° until all skins have been removed. It is essential that the edges of each skin be flattened out, otherwise the skin may not float properly. Using forceps, each skin is carefully floated on the surface of an 0.25% trypsin solution (w/v) at 4°, with the epidermis facing upward, for 15-24 hr. Skins are individually transferred to a sterile surface and stretched with the epidermis facing down, and the epidermis is separated from the dermis using forceps, minced, and stirred in standard medium (see below), with the calcium adjusted to 1.4 raM, to release keratinocytes. To remove cornified sheets, the cell suspension is 27 M, Reiss, V. F. Vellucci, and Z. L. Zhou, Cancer Res. 53, 899 (1993).

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[1] Hypodermis

Derm~rmis

DorsalSkin Epidermis

[ Scrape Off Epidermal Cells with Forceps

Dermis FloatSkin for 1 Hour ~ FilterP a p e r

~ 37°

1% Trypsin

FilterPaper i:.:.:.:.:.:.:.:.:.:.:.:.:.1 F+:+:':':+:':':':':':':+:+:':+:+:':I

Sterile Surface

Dermis Epidermis

F ~ . 2. Isolation of primary keratinocytes from adult mouse skin. After scraping keratinocytes off the dermis with forceps, cells are resuspended in standard medium, with Ca 2÷ adjusted to 1.4 mM, and filtered through nylon gauze as in Fig. 1. See text for additional details.

filtered through a sterile, 100-t~m mesh nylon gauze (Nytex 157 mesh, Martin Supply Co., Baltimore, MD), and cells are plated at a density of 2-4 × 105/cmz in standard culture medium with Ca 2+ adjusted to 0.1-0.3 mM. After 16-24 hr, cells are washed with Ca 2+- and Mg2+-free phosphatebuffered saline (PBS), and switched to standard medium containing 0.05 mM Ca 2+ (see below). 28 When epidermis from transgenic mice is utilized, intact skins can be stored overnight at 4° in standard medium containing 1.4 mM Ca 2+, while genotyping is being performed. Adult mouse epidermal cells may be isolated from two sources: haired dorsal skin and hairless tail and ear skin. For dorsal skin, mice must be in the resting phase of the hair cycle, which can be determined by the absence of hair growth for 24-48 hr after clipping. Adult mice are sacrificed by COz narcosis and dorsal skin is depilitated with Nair (Carter Products Inc., New York, NY). The entire mouse is washed in Betadine and 70% ethanol as described earlier. Dorsal and ventral skin is removed surgically and placed epidermis side down on a sterile surface (Fig. 2). The hypodermis is removed by vigorous scraping with a scalpel--a procedure essential for the successful separation of epidermis from dermis. The skin is cut into 3to 4-cm 2 pieces, and autoclaved filter paper (Whatman No. 1, Wardstone, England) is placed on the exposed surface of the dermis as a support. The skin and filter paper are inverted onto the surface of 1% trypsin in PBS (2.5% trypsin stock, Bio-Whittaker, Walkersville, MD, Cat. No 17-160H) for 1 hr at 37°. Skins are then removed to a dry sterile surface, the epidermis 28 H. Hennings, D. Michael, C. Cheng, P. Steinert, K. Holbrook, and S. H. Yuspa, Cell (Cambridge, Mass.) 19, 245 (1980).

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is scraped off the dermis with curved forceps, and the keratinocytes are filtered and resuspended as for newborn skin. The approximate yield from dorsal adult mouse skin is 5-10 x 106 cells/skin. Alternatively, nonhairy tail or ear skin is washed in Betadine and 70% ethanol. 29 A longitudinal incision is made through the tail length and skin is removed using forceps. The two skin flaps composing the ear must be separated carefully. The trypsinization of tail or ear skin requires 1.5 hr of flotation on 1% trypsin at 37 ° and yields approximately 10 × 106 keratinocytes per tail and 6 × 10 6 per pair of adult ears. Adult keratinocytes do not attach well to culture dishes, and coating of plastic dishes with 30 tzg/ml of collagen type I (Vitrogen 100, Celtrix Laboratory, Palo Alto, CA, Cat. No. PC0701), collagen type IV, or fibronectin is essential for the proper attachment and spreading of primary cells. To achieve maximal attachment and growth of adult and newborn keratinocytes, dishes with a keratinocyte extracellular matrix can be prepared from confluent cultures of newborn keratinocytes. Culture dishes previously plated with newborn keratinocytes are incubated with 0.025 M NH4OH/0.5% Triton X-100 (v/v) in PBS for 1-5 min at room temperature, followed by several washes with I m M E D T A in PBS. Dishes can be stored with PBS at 4 ° for at least 3-4 weeks until used for the plating of freshly isolated keratinocytes.

Human

Keratinocytes

H u m a n keratinocytes can be prepared from fetal, adult, and newborn sources. 3°'31 Newborn foreskin is a common source. The foreskin is spread, and the muscular and mucosal layers are completely removed from the hypodermis by scraping vigorously with a scalpel. The tissue is floated on 0.25% trypsin at 4 ° for 18-24 hr. The epidermis is then separated from the dermis by forceps, minced, and stirred in medium. The dissociated single cell suspension is filtered through Nytex gauze and centrifuged, and pelleted cells are resuspended and plated. Each foreskin yields 2 - 4 x 106 viable cells. Skin tissue specimens can also be obtained from surgical procedures, amputations, or suction blisters--a routine procedure used for allografts and autografts in clinical applications. 32 When small biopsies are obtained, floating on 0.25% trypsin for 1-2 hr at 37 ° is sufficient for the proper separation of epidermis from dermis. The development of defined medium 29T. Tennenbaum, S. H. Yuspa, and J. Kapitulnik, J. Cell. Physiol. 143, 431 (1990). ~0j. G. Rheinwald and H. Green, Cell (Cambridge, Mass.) 6, 331 (1975). ~ A. R. Haake and A. T. Lane, In Vitro Cell Dev. BioL 25, 592 (1989). 32M. R. Pittelkow and R. E. Scott, Mayo Clin. Proc. 61,771 (1986).

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for optimal growth of human keratinocytes enables the growth of cells at clonal densities, prolongs the life span, and increases the growth rate without the need of 3T3 feeder layers (see below). Primary human keratinocyte cultures can be subcultured repeatedly using a dilute t r y p s i n / E D T A solution, 0.1%/0.02%, in PBS. In an effort to develop a cultured skin system that will mimic the characteristics of normal skin, cultures of primary keratinocytes are layered on a dermal equivalent. Dermis-like structures can be composed of organic deepidermized dermis kept at - 8 0 ° until use. 33 Alternatively, collagen gels or a combination of collagen lattices and fibroblasts can be utilized as collagen gels. 34 The mixture of collagen, fibroblast, and complete culture medium is cast in petri dishes. After 3 days, gelation and contraction of the dermal equivalent are completed, and keratinocytes are layered on top at the a i r - m e d i u m interface, resulting in a three-dimensional organ culture system. 34 These reconstituted skin equivalents stratify and differentiate similarly to normal skin with compartmentalized expression of markers specific to the different epidermal strata and polarized expression of basement membrane components. 34 This system enhances the ability to study the invasive characteristics of oncogene-transduced human keratinocytes and considers the influence of oncogenes on keratinocyte-fibroblast interactions. 21 This model was also reported to support the propagation of oncogenic human papilloma virus in vitroJ 5 Culture Media In early studies, keratinocytes derived from mouse epidermis grew poorly in vitro. The discovery that Ca 2+ is a key regulator of keratinocyte differentiation enabled the development of improved culture media. Proliferation of murine epidermal keratinocytes in vitro requires an extracellular Ca 2+ concentration <0.10 raM, with higher Ca 2+ levels inducing terminal differentiation. 28'36Since serum is required for the optimal growth of murine keratinocytes, it must first be depleted of Ca 2+ using either a chelating resin or dialysis. Fetal bovine serum (FBS) is passed through a column of Chelex 100 chelating resin (200-400 mesh, sodium form, Bio-Rad Laboratories, 33 N. Basset-Seguin, J. F. Culard, C. Kerai, F. Bernard, A. Watrin, J. Demaille, and J. J. Guilhou, Differentiation (Berlin) 44, 232 (1990).

34E. Bell, S. Sher, B. Hull, C. Merrill,S. Rosen, A. Chamson, D. Asselineau, and L. Dubertret, J. Invest. Dermatol. 81, 2s (1982). 35S. C. Dollard, J. L. Wilson, L. M. Demeter, W. Bonnez, R. C. Reichman, T. R. Broker, and L. T. Chow, Genes Develop. 6, 1131 (1992). 36S. H. Yuspa, A. E. Kilkenny,P. M. Steinert, and D. R. Roop, J. Cell Biol. 109, 1207 (1989).

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Richmond, CA, Cat. No. 142-2842), followed by 0.2/xm filter sterilization. Standard growth medium is prepared using Ca2+-free Eagle's minimum essential medium (EMEM) with Earle's balanced salt solution, nonessential amino acids, and L-glutamine (Bio-Whittaker, Cat. No. 06-174D), supplemented with 8% Chelex-treated FBS, 0.25% penicillin-streptomycin solution (10,000 units/ml penicillin G sodium and 10 mg/ml streptomycin sulfate in 0.85% saline, GIBCO, Grand Island, NY, Cat. No. 600-514OAG), and the appropriate volume of CaC12 (280 mM stock) for a final concentration of 0.05 mM Ca 2+. The amount of CaC12 is determined for each batch of Chelex-treated serum by measuring the residual Ca 2+ concentration in complete standard growth medium using atomic absorption spectrophotometry and adding sufficient CaC12 to a final concentration of 0.05 mM. Serum lots should be screened due to variability in supporting keratinocyte growth. When grown in standard medium and replenished three times a week, primary murine keratinocytes can be maintained as a proliferating monolayer for up to 2 weeks. In general, these cultures cannot be successfully passaged. Mouse epidermal keratinocytes can also be grown in a serumfree medium containing multiple defined supplements and bovine pituitary extract. 37 This medium has been reported to support clonal growth, longterm cultivation (25-30 population doublings), and repeated subculturing of murine keratinocytes.37 Another serum-free nutrient-enriched medium has been developed for use with murine keratinocytes which does not require reduced levels of extracellular Ca2+.3s Successful long-term cultivation of human keratinocytes was first achieved by growing cells on a feeder layer of irradiated mouse 3T3 fibroblasts in medium supplemented with 20% serum, hydrocortisone, and epidermal growth factor (EGF). 3° Serum-free media have subsequently been developed which support the growth of human keratinocytes for several passages, without the requirement of a feeder layer. Most of these are modifications of a formulation (MCDB 153) originally developed by Boyce and Ham, 39 containing reduced levels of extracellular Ca 2+ and several additives. Cells derived from newborn or adult human skin are routinely grown in an enriched MCDB 153 medium containing 74 ng/ml hydrocortisone, 5/xg/ml insulin, 6.7 ng/ml triiodothyronine, 5 ng/ml EGF, 50 txg/ml bovine pituitary extract, and 0.09 mM Ca 2+ (Keratinocyte-SFM, GIBCO BRL, Gaithersburg, MD, Cat. No. 320-7005PJ). Clonetics Corporation (San 37F. Bertolero, M. E. Kaighn,M. A. Gonda, and U. Saffiotti,Exp. Cell Res. 155,64 (1984). 3sR. J. Morris, K. C. Tacker,J. K. Baldwin,S. M. Fischer, and T. J. Slaga, Cancer Lett. 34, 297 (1987). 3~S. T. Boyce and R. G. Ham,J. Invest. Dermatol. 81, 33s (1983).

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Diego, CA) offers a MCDB 153-based medium which can also be used, designated KGM (Cat. No. CC-3001). Primary human keratinocytes can be passaged several times when grown in these media.

Transfection and Selection Methods

Introduction of Foreign DNA Foreign DNA can be introduced into keratinocytes using several methods, including calcium phosphate- and lipid-mediated transfection and retroviral infection. Because of the differentiating effect of extracellular Ca 2+ on keratinocyte cultures, 2s the calcium phosphate method has been modified.4° Murine cultures are maintained in standard 0.05 mM Ca z+ growth medium (see earlier), and culture medium is changed 1 day prior to transfection. Four hours prior to transfection, cultures are washed twice with PBS and incubated with 4.5 ml of standard growth medium containing 0.01 mM K + (prepared from K +- and CaZ+-free EMEM, Bio-Whittaker, Cat. No. 04544D) per 60-mm dish of cells. DNA is diluted in 1 ml 0.25 M CaCI2 and is precipitated with 1 ml of 2× HEPES-buffered saline, pH 7.04 (1 ml of 1 M HEPES, pH 7.04, 30/xl of 1 M NaHPO4, and 2.5 ml of 2.5 M NaCI in 5 mM HEPES, pH 7.3, in 20 ml total volume), adding 3 drops at a time with gentle agitation. Precipitate (0.9 ml/60-mm dish) is added, and the dish is rotated to evenly distribute the precipitate. After an additional 4 hr, the cells are washed once with 3 ml PBS, treated for 3 rain with 2 ml of 25% dimethyl sulfoxide (DMSO) in 0.01 mM K + medium, washed three times with 5 ml PBS, and incubated for 16 hr in fresh 0.01 mM K + medium before washing and replenishing with standard medium. Up to three plasmid constructs have been introduced in combination41; a total of 20/zg/60-mm dish of CsCl-purified plasmid is added using sheared salmon sperm DNA or noncoding plasmid vector as carrier DNA. Transfection of DNA for transient gene expression can be performed on primary cultures from newborn or adult mice within the first 6 days of culture with similar results. Cell lines are plated in standard medium (1-2 × 106/60-mm dish) 1-2 days prior to transfection. Several lipid-based methods of transfection have become available. These methods require less plasmid and are less laborious. Murine keratinocytes are washed once with serum-, Ca 2+-, and antibiotic-free EMEM and are covered with 2 ml of a lipid-DNA complex/60-mm dish for 6 hr [2 /zg DNA and 6/zl LipofectAMINE (GIBCO BRL, Cat. No. 18324-012) 4o j. R. Harper, D. A. Greenhalgh, and S. H. Yuspa, J. Invest. DermatoL 91, 150 (1988). 41 T. Kartasova, D. R. Roop, K. A. Holbrook, and S. H. Yuspa, J. Cell Biol. 120, 1251 (1993).

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preincubated for 30-45 min in standard medium without serum or antibiotic], then washed and allowed to recover in standard medium for at least 12 hr before further experimentation. Best results are obtained if primary cultures are confluent at the time of lipofectamine-mediated transfection. Other liposome-mediated protocols have also been successfully applied for human keratinocyte transfections. 42 Both stable and transient gene expression can also be achieved following electroporation of human and mouse keratinocyte cell suspensions. 24,43'44 Human foreskin keratinocytes can be efficiently transfected with lipid- and calcium phosphate-mediated methods, but Polybrene-mediated transfection is recommended. 44 D N A (0.5-3/~g) is added to keratinocytes in the presence of 10-20/xg/ml Polybrene to increase the adsorption of D N A to the cell surface. After 6 hr, the DNA/Polybrene mix is removed, the uptake of adsorbed D N A is facilitated with a 3-min treatment with 25-30% DMSO, and fresh culture medium is added. 44,45 Because of the higher efficiency of viral infection, retroviral vectors are better suited for establishing mass cultures of primary keratinocytes expressing exogenous DNA. 46 A replication-defective retrovirus restricts genetransfer to cultured cells and therefore simplifies the evaluation of in vivo tests of recipient cells. Moloney-based retroviral systems have been successfully employed for transducing D N A into keratinocytes. 47'4s Cultured mouse keratinocytes are washed with PBS and incubated with virus, at a multiplicity of infection of 1 : 1, in a total volume of 0.5 ml/60-mm dish in standard medium in the presence of 4/zg/ml Polybrene, tilting culture trays every 15 min to ensure that the cells remain moist. 47 After 1-1.5 hr. 2.5 ml of standard medium is added and replaced with fresh virus-free standard medium after 48-72 hr. Hair follicles can be infected in suspension following the same procedure but in a 50-ml tube, swirling every 15 min. 4'~ Murine keratinocytes can be infected with multiple retroviruses in combination. Human keratinocytes can be transduced by retroviral vectors using a

42 X. F. Pei, J. M. Meck, D. Greenhalgh, and R. Schlegel, Virology 196, 855 (1993). 4~ M. Reiss, M. M. Jastreboff, J. R. Bertino, and R. Narayanan, Biochem. Biophys. Res. Commun. 137, 244 (1986). 44 C. K. Jiang, D. Connolly, and M. Blumenberg, J. Invest. Dermatol. 97, 969 (1991). 45 j. S. Rhim, J. B. Park, and G. Jay, Oncogene 4, 1403 (1989). 46 j. A. Garlick, A. B. Katz, E. S. Fenjves, and L. B. Taichman, J. Invest. Dermatol. 97, 824 (1991). 47 D. R. Roop, D. R. Lowy, P. E. Tambourin, J. Strickland, J. R. Harper, M. Balaschak. E. F. Spangler, and S. H. Yuspa, Nature (London) 323, 822 (1986). 48 O. Danos and R. C. Mulligan, Proc. Natl. Acad. Sci. U.S.A. 85, 6460 (1988). 49 W. C. Weinberg, D. Morgan, C. George, and S. H. Yuspa, Carcinogenesis (London) 12, 1119 (1991).

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similar procedure, with Polybrene concentrations differing between investigators. 46,50 P l a s m i d Choice and Selection o f Stably Expressing Cells

M a n y constitutively active, virally derived p r o m o t e r s are highly expressed in h u m a n and mouse keratinocyte cultures, including those from CMV, SV40, and RSV41'44,51; however, expression driven by viral promoters is c o m m o n l y modulated by extracellular Ca2+. 51 A genomic p r o m o t e r sequence has b e e n defined within the mouse c-ras Ha gene which demonstrates strong p r o m o t e r activity in murine keratinocytes with less Ca 2+ responsiveness. 52 Plasmid constructs encoding specific exogenous sequences regulated by the metallothionine gene p r o m o t e r have also been used in mouse keratinocytes. 41 P r o m o t e r sequences from genes expressed specifically according to the differentiation state which have been utilized in mouse cell transfections include the 6000-bp p r o m o t e r sequence of the h u m a n keratin 5 gene 53 and Ca2+-inducible regulatory elements 3' to the h u m a n keratin 1 coding sequence. 54 Regulatory sequences from the h u m a n keratin 1, bovine keratin 10, and h u m a n keratin 14 genes have been used to target the expression of several oncogenes and growth factors or their receptors to specific subpopulations within the epidermis of transgenic m i c e Y -57 For stable transfected keratinoeyte cell lines, commonly used selectable markers include the genes for neomycin and hygromycin resistance in a 1 : 10 ratio with the sequence of interest. Stable transfectants are difficult to isolate from primary cultures of mouse keratinocytes. Primary mouse keratinocytes are very sensitive to these selecting agents (6/xg/ml G418, 0.5/xg/ml hygromycin), and resistant cells grow poorly at clonal density. G r e a t e r success has b e e n achieved with established murine keratinocyte cell lines, which require from 40 to 200 /zg/ml G418 and 3 to 8 /zg/ml hygromycin to eliminate nonrecipients of these selectable markers.

.~0C. D. Woodworth, H. Wang, S. Simpson, L. M. Alvarez-Salas, and V. Notario, Cell Growth Differ. 4, 367 (1993). 5aj. I. Lee and L. B. Taichman, J. Invest. Dermatol. 92, 267 (1989). 52R. Neades, N. A. Betz, X. Y. Sheng, and J. C. Pelling, Mol. Carcinog. 4, 369 (1991). 53C. Byrne and E. Fuchs, Mol. Cell. Biol. 13, 3176 (1993). 54C. A. Huff, S. H. Yuspa, and D. Rosenthal, J. Biol. Chem. 268, 377 (1993). 55D. A. Greenhalgh, J. A. Rothnagel, X. J. Wang, M. I. Quintanilla, C. C. Orengo, T. A. Gagne, D. S. Bundman, M. A. Longley, C. Fisher, and D. R. Roop, Oncogene 8, 2145 (1993). 56S. Werner, W. Weinberg, X. Liao, K. G. Peters, M. Blessing, S. H. Yuspa, R. L. Weiner, and L. T. Williams, EMBO J. 12, 2635 (1993). 57R. Vassar, M. E. Hutton, and E. Fuchs, Mol. Cell. Biol. 12, 4643 (1992).

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T r a n s f e c t e d h u m a n k e r a t i n o c y t e s r e q u i r e 1 0 0 - 8 0 0 / x g / m l G41821'5859 a n d 2 0 / x g / m l h y g r o m y c i n 24 for effective selection.

M a r k e r s of N e o p l a s t i c T r a n s f o r m a t i o n In Vitro M a r k e r s

I n cultures of n o r m a l m o u s e k e r a t i n o c y t e s , raising extracellular Ca 2+ f r o m 0.05 to > 0 . 1 0 m M triggers irreversible g r o w t h - a r r e s t c o u p l e d to the e x p r e s s i o n of m u l t i p l e e p i d e r m a l d i f f e r e n t i a t i o n m a r k e r s . Ca 2+ also i n d u c e s d i f f e r e n t i a t i o n of h u m a n k e r a t i n o c y t e s at higher c o n c e n t r a t i o n s ( 0 . 3 - 2 m M Ca2+). I n d u c t i o n of d i f f e r e n t i a t i o n m a r k e r s in r e s p o n s e to Ca 2+ is typically d e l a y e d or i n h i b i t e d in k e r a t i n o c y t e s t r a n s d u c e d with c e r t a i n o n c o g e n e s ; m o r e o v e r , in s o m e cases, m a r k e r s n o t n o r m a l l y expressed in e p i d e r m a l cells are i n d u c e d a b e r r a n t l y ( T a b l e I)60-75 I n addition, f o r m a t i o n of a crossl i n k e d cornified cell e n v e l o p e a n d d e t a c h m e n t of cells from the culture s u b s t r a t u m , which m a r k the final stage of s q u a m o u s d i f f e r e n t i a t i o n , are characteristically i n h i b i t e d in n e o p l a s t i c k e r a t i n o c y t e s ( T a b l e I). T h e acq u i r e d resistance to Ca2+-mediated d i f f e r e n t i a t i o n has b e e n used to select for o n c o g e n e - a l t e r e d k e r a t i n o c y t e s in vitro. O n c o g e n e s are i n t r o d u c e d into cultures of p r o l i f e r a t i n g k e r a t i n o c y t e s using t e c h n i q u e s o u t l i n e d earlier. ss C. D. Woodworth, S. Cheng, S. Simpson, L. Hamacher, L. T. Chow, T. R. Broker, and J. A. DiPaolo, Oncogene 7, 619 (1992). s9 L. Pirisi, S. Yasumoto, M. Feller, J. Doniger, and J. A. DiPaolo, J. Virol. 61, 1061 (1987). ~,0C. Cheng, A. E. Kilkenny, D. Roop, and S. H. Yuspa, Mol. Carcinog. 3, 363 (1990). ~'~M. Reiss, D. DiMaio, and T. A. Zibello, Cancer Commun. 1, 75 (1989). 62T. S. Hronis, M. L. Steinberg, V. Defendi, and T. T. Sun, Cancer Res. 44, 5797 (1984). ~3N. Sheibani, J. S. Rhim, and B. L. Allen-Hoffmann, Cancer Res. 51, 5967 (1991). ~4X. F. Pei, P. A. Gorman, and F. M. Watt, Carcinogenesis (London) 12, 277 (1991). ~5C. Missero, C. Serra, K. Stenn, and G. P. Dotto, J. Cell Biol. 121, 1109 (1993). ~'¢'C. Missero, E. Filvaroff, and G. P. Dotto, Proc. Natl. Acad. Sci. U.S.A. 88, 3489 (1991). ~,7M. Diaz-Guerra, S. Haddow, C. Bauluz, J. L. Jorcano. A. Cano, A. Balmain, and M. Quintanilla, Cancer Res. 52, 680 (1992). 6~D. R. Henrard, A. T. Thornley, M. L. Brown, and J. G. Rheinwald, Oncogene 5,475 (1990). 6'*X. F. Pei, I. M. Leigh, and F. M. Watt, Epithelial Cell Biol. 1, 84 (1992). 7oL. Pirisi, K. E. Creek, J. Doniger, and J. A. DiPaolo, Carcinogenesis (London) 9,1573 (1988). 71 p. Kaur and J. K. McDougall, J. Virol. 62, 1917 (1988). 72M. Darmon, C. Delescluse, A. Semat, B. Bernard, J. Bailly, and M. Prunieras, Exp. Cell Res. 154, 315 (1984). 73j. Taylor-Papadimitriou, P. Purkis, E. B. Lane, I. A. McKay, and S. E. Chang, Cell D(£fer. 11, 169 (1982). 74C. Agarwal and R. L. Eckert, Cancer Res. 50, 5947 (1990). 7s E. K. Parkinson, P. Grabham, and A. Emmerson, Carcinogenesis (London) 4, 857 (1983).

14

[ 1]

CELLS TABLE I

ONCOGENE-INDUCED PHENOTYPIC ALTERATIONS IN CULTURED EPIDERMAL KERATINOCYTES

Differentiation marker(s)

Oncogene

Species

Reduced or Delayed Expression of Differentiation Markers Keratins K1 and 10 Mouse BPV 1 Transglutaminase Mouse a HPV 18 E6/E7 K1, involucrin Human EBV-LMP, EJ r a s Ha Involucrin Human b SV40 Involucrin Human HPV 16 Involucrin Human E1A Transglutaminase Mouse E1A Transglutaminase Mouse c v - r a s Ha

v - r a s Ha

T24

c - r a s Ha

v - r a s Ha

HPV 16 HPV 18 SV40 SV40 large T A g

Oncogene v - r a s Ha, v - r a s Ki

HPV 16 HPV 16, 18 SV40 SV40 HPV 18 EBV-LMP, EJ

r a s Ha

Refs.

60 61 58 13 62 63, 64 65 66

Appearance of "Aberrant" Keratins K8, K18 Mouse K8 K18 Mouse d K19 Human K18, K19 Human K19 Human K8, K18, K19 Human K7 Human

60 67 68 69, 70 71 62, 72, 73 74

Inhibition of Squamous Differentiation Differentiation stimulus Species

Refs.

Ca 2+ Suspension Ca 2+ + serum TPA Ca 2+ ionophore Ca 2÷, TPA Ca z÷ ionophore

Mouse Human Human Human Human Human Human h

23 63 26 75 2 71 13

BALB/MK cell line. h SCC12F cell line. c PAM212 cell line. d MCA3D cell line; other studies used nonimmortalized, early passage epidermal keratinocytes.

When cultures are subsequently grown in medium with an increased Ca 2+ level to induce terminal differentiation,"Ca2+-resistant '' loci emerge, representing the clonal expansion of single, oncogene-transduced keratinocytes (Table I). Oncogene-transduced keratinocytes are also resistant to other differentiation stimuli, including the phorbol ester TPA, Ca 2+ ionophores, and growth in suspension (Table I). In addition to alterations in their ability to terminally differentiate, cultured keratinocytes expressing a variety of

[1]

KERATINOCYTES

15

different oncogenes exhibit abnormalities in growth regulation (Table I I ) . 76-s3

Growth in a semisolid medium is a sensitive assay in testing the anchorage-independent growth of malignant fibroblasts. However, in cultures of human and mouse keratinocytes, growth in suspension may induce the differentiation of malignant cell lines 63"84as well as normal keratinocytes, and growth in semisolid medium generally represents a later stage of malignant conversion or invasionY ,86 Keratinocytes (usually 3 × 105) are plated in complete medium (MCDB 153 for human and EMEM for mouse keratinocytes) containing 1.5% methylcellulose with standard Ca 2+ concentrations. Cells are incubated at 37 ° for 4 days. The suspension is diluted in complete medium and plated on 60-mm dishes. The dishes are kept in culture for 10 days and subsequently are fixed with 3% formaldehyde and stained with Giemsa. Colonies with more than 16 cells are countedY In Vivo Markers

Two types of in vivo systems have been employed to analyze the tumorigenic properties of keratinocyte recipients of specific oncogenes: (1) subcutaneous injections and (2) skin grafting of the test cells onto the dorsal epidermis of an adult athymic or syngeneic adult or newborn mouse host (Table III). s7-93 With subcutaneous injections, only epithelial cells are intro76 M. L. Steinberg and V. Defendi, Proc. NatL Acad. Sci. U.S.A. 76, 801 (1979). 77 y. Barrandon, J. R. Morgan, R. C. Mulligan, and H. Green, Proc. Natl. Acad. Sci. U.S.A. 86, 4102 (1989). 7s M. Durst, R. T. Dzarlieva-Petrusevska, P. Boukamp, N. E. Fusenig, and L. Gissmann, Oncogene 1, 251 (1987) 79 M. W. Appleby, I. M. Greenfield, T. Crook, E. K. Parkinson, and M. A. Stanely, Oncogene 4, 1323 (1989). s, j. p. Falco, W. G. Taylor, P. P. DiFiore, B. E. Weissman, and S. A. Aaronson, Oncogene 2, 573 (1988). sl A. B. Glick, M. B. Sporn, and S. H. Yuspa, Mol. Carcinog. 4, 210 (1991). s2 j. A. Pietenpol, R. W. Stein, E. Moran, P. Yaciuk, R. Schlegel, R. M. Lyons, M. R. Pittelkow, K. Munger, P. M. Howley, and H. L. Moses, Cell (Cambridge, Mass.) 61, 777 (1990). s3 M. Sebag, J. Henderson, J. Rhim, and R. Kremer, J. Biol. Chem. 267, 12162 (1992). sa N. E. Fusenig, S. M. Amer, P. Boukamp, and P. K. Worst, Bull. Cancer 65, 271 (1978). s5 j. G. Rheinwald and M. A. Beckett, Cell (Cambridge, Mass.) 22, 629 (1980). s6 N. H. Colburn, W. F. Vorder Bruegge, J. R. Bates, R. H. Gray, J. D. Rossen, W. H. Kelsey. and T. Shimada, Cancer Res. 38, 624 (1978). s7 G. P. Dotto, R. A. Weinberg, and A. Ariza, Proc. Natl. Acad. Sci. U.S.A. 85, 6389 (1988). ss J. L. Brissette, C. Missero, S. H. Yuspa, and G. P. Dotto, Mol. Carcinog. 7, 21 (1993). s9 D. A. Greenhalgh, D. J. Welty, A. Player, and S. H. Yuspa, Proc. Natl. Acad. Sci. U.S.A. 87, 643 (1990). 9~ M. S. Lee, J. H. Yang, Z. Salehi, P. Arnstein, L. S. Chen, G. Jay, and J. S. Rhim, Oncogene 8, 387 (1993).

16

CELLS

[1l

TABLE II ONCOGENE-INDUCED ALTERATIONS IN GROWTH REGULATION OF EPIDERMAL KERATINOCYTES in Vitro

Oncogene

Effect

Species

Refs.

SV40 E1A HPV 16 HPV 18 HPV 18 E6/E7 Adl2-SV40 Polyomavirus large T A g v-fos, EJ ras na SV40 HPV 16 v-fos, EJ ras Ha

Immortalization Immortalization Immortalization Immortalization Immortalization Immortalization Immortalization Immortalization Reduced serum requirement Reduced serum requirement Reduced serum requirement EGF-independent

Human Human Human Human Human Human Rat Mouse Human Human Mouse Mouse a

76 77 59, 78 71 24 15 9 79 2, 76 70 79 80

EGF/insulin-independent EGF-independent TGFc~ expression increased Hyperproliferation Less responsive to growth inhibition by TGF-/3 Less responsive to growth inhibition by TGF-/3 Less responsive to growth inhibition by TGF-/3 Less responsive to growth inhibition by vitamin D 3

Mouse a Human Mouse Rat c Human

80 68 81 10 82

Mouse a

66

Mouse a

27

Human e

83

v-ras Ha, v-ras Ki, v-mos, v-erbB, v-fins v-fgr v-ras Ha v-ras Ha c-ras Ha (protooncogene) b

HPV 16, 18; SV40 E1A p53 (mutant) ras Ha

a BALB/MK keratinocyte cell line cultured in a defined medium. b Overexpressed. c FRSK cell line. a PAM212 cell line. e HPK1A cell line.

duced. Approximately 106-107 cells are injected in 0.1 ml PBS into the interscapular region of the h o s t . 19 Most malignant keratinocytes can grow subcutaneously, whereas papilloma or benign tumor cells do not form tumors 94 (Table III). 91 D. A. Greenhalgh and S. H. Yuspa, Mol. Carcinog. 1, 134 (1988). 92 C. M. Kim, J. Vogel, G. Jay, and J. S. Rhim, Oncogene 7, 1525 (1992). 93 E. Finzi, A. Kilkenny, J. E. Strickland, M. Balaschak, T. Bringman, R. Derynck, S. Aaronson, and S. H. Yuspa, Mol. Carcinog. 1, 7 (1988). 94 j. E. Strickland, D. A. Greenhalgh, A. Koceva-Chyla, H. Hennings, C. Restrepo, M. Balaschak, and S. H. Yuspa, Cancer Res. 48, 165 (1988).

[ 1]

KERATINOCYTES

17

TABLE III In Vivo EFFECTS OF ONCOGENES ON KERATINOCYTES

Oncogene

Keratinocyte recipient Primary

Species

Transplant type

Ela c-myc p53mut

RHEK-I" PA-PE ~' p117 h Primary RHEK-1 308/SP1 ~ 308/SP1 308/SP1 p 117

Mouse Mouse Human Mouse Mouse Mouse Human Mouse Mouse Mouse Mouse

Graft Graft Subcutaneous Graft Graft Graft Subcutaneous Graft Graft Graft Graft

neu

pl 17

Mouse

Graft

fes, ¢ms erbB, src HIVtat TGF-a v-fos/v-ras v-fos/HPV18 HPV HPV

RHEK-1

Human

Subcutaneous

RHEK-1 308/SP1 Primary Primary Primary Primary

Human Mouse Mouse Human Human Human

SV40 HHV-6

Primary RHEK-1

Human Human

Subcutaneous Graft Graft Subcutaneous Subcutaneous Subcutaneous graft under skin flap J Subcutaneous Subcutaneous

ras

v-fos

Phenotype Papilloma Carcinoma' Carcinoma Carcinoma Carcinoma Normal skin Carcinoma Carcinoma Papilloma Papilloma Dysplastic papilloma Dysplastic papilloma Carcinoma

Refs. 47 87, 88 15 20 21 89 90 91 91 91 21 2l 3

Carcinoma Papilloma Carcinoma Carcinoma Nontumorigenic Dysplastic Epithelium

92 93 89 42 25.70. 71 4

Nontumorigenic Carcinoma

2 12

"Continuous human cell line, contains Adl2-SV40. t' Papilloma cell lines, contain activated c-rasnL ' Phenotype dependent on high v-ras Ha expression. d Cultured epithelial sheet implanted intact between dorsal musculature and skin.

Skin reconstitution or grafting is a more widely used method since it has the advantage of allowing the study of cells at intermediate stages of premalignant progression. In some studies the graft site on the host is prepared by inserting a ground glass disk (26 x 3 mm) between the skin and thoracic wall to induce a capsule of granulation tissue, s4 The disk is first inserted at an incision at the base of the tail and moved up to the center of the back. After 3 - 4 weeks the disk is removed and a silicone grafting chamber is held in place using surgical clips. The grafting chamber (Renner G M B H , 6701 Dannstadt-Schauerheim 1, Riedstrasse 6 Germany) consists of an upper hat-shaped dome with a central 3-mm hole and a lower

18

CELLS

[ 1]

Ft~. 3. Schematic cross section of silicone grafting chamber held in place under dorsal skin with surgical clips.

chamber which fits into the dome that is an open cylinder with a broad rim 95 (Fig. 3). More recent studies have shown that the granulation bed is not required for successful transplantation. 95'96In this method the assembled chamber is put in place immediately before adding the test cells. The dorsal epidermis of anesthetized mice is wiped with Betadine and 70% ethanol. An approximately 1-cm-diameter circle of dorsal epidermis is removed, using curved scissors, and the wound is treated with an antibiotic spray (Polysporin, Burroughs-Wellcome, Research Triangle Park, NC). The assembled grafting chamber is inserted directly, with the rim of the chamber underneath the host skin, and is held in place with surgical clips. In both methods the graft chambers are removed 1 week after the addition of the transplanted cells, and the grafts are allowed to heal. Two types of cell preparation techniques have been employed: (1) transplantation of an organotypic culture directly to the graft site, or (2) transplantation of suspensions of keratinocytes and fibroblasts. In the first method, either mouse or human keratinocytes are plated in petri dishes on a thin collagen gel prepared within the lower part of the grafting chamber. Dermal fibroblasts can also be plated directly into the collagen gel prior to plating the keratinocytes. After 2 days in culture, the top of the grafting chamber is added and the culture is transplanted to the h o s t . 97 Alternatively, 95 j. E. Strickland, A. A. Dlugosz, H. Hennings, and S. H. Yuspa, Carcinogenesis (London) 14, 205 (1993). 96 W. C. Weinberg, L. V. Goodman, C. George, D. L. Morgan, S. Ledbetter, S. H. Yuspa, and U. Lichti, J. Invest. Dermatol. 100, 229 (1993). 97 m. Bohnert, J. Hornung, I. C. Mackenzie, and N. E. Fusenig, Cell Tissue Res. 244, 413 (1986).

[ 1]

KERATINOCYTES

19

suspensions of epidermal cell lines or primary epidermal cultures and primary dermal fibroblasts96 can be combined, pelleted, and the thick slurry of cells added directly to the grafting chamber in place on the host, through the hole in the top dome. 95 Routinely 5-6 × 106 epidermal cells are combined with 8 × 10 6 fibroblasts. It is important to use dermal fibroblasts that have been cultured for at least 1 week to avoid generation of hair in the graft 96 and to remove any epithelial cells that could modulate the behavior of the test cells. Aseptic techniques must be used at all times in preparation of cells and during grafting since transplanted cells usually do not survive if the graft site becomes infected. Growth of transplanted tumor cells is usually apparent by 1 week after dome removal. In addition to histological characterization, graft tumors can be analyzed using a series of markers that are associated with malignant progression of epidermal tumors. Tumors generated from oncogene-transduced grafted mouse keratinocytes have a similar pattern of marker expression as chemically induced mouse skin tumors. 98 Specific polyclonal antibodies have been developed which allow in situ localization of protein markers. 99 Keratins 1 and 10 are expressed in normal epidermis and well-differentiated papillomas, whereas keratin 13 is expressed during premalignant progression, often in cells that have lost expression of keratins 1 and 10. t°° Keratin 8 is expressed only in highly dysplastic papillomas and in squamous carcinomas. 1°°'1°1 Additionally, the c~6/34 integrin is a useful marker for the basal compartment which expands during the premalignant progression of mouse papillomas, t°° TGF-/31 and TGF-/32 immunostaining is also lost during the premalignant progression of chemically induced and grafted mouse skin tumorsJ °2 Human tumors also have a similar loss of keratins 1 and 10, and appearance of keratin 8. l°3 However, expression of markers in grafted oncogene-transduced keratinocytes has not been characterized. In general, sections from tissues frozen in OCT (Miles, Elkhart, IN) give the best results in immunohistochemistry, although Carnoy's fixative (60% ethanol:30% 9s T. Tennenbaum, S. H. Yuspa, A. Grover, V. Castronovo, M. E. Sobel, Y. Yamada, and L. M. De Luca, Cancer Res. 52, 2966 (1992). '~'~D. R. Roop, C. K. Cheng, L. Titterington, C. A. Meyers, J. R. Stanley, P. M. Steinert, and S. H. Yuspa, J. Biol. Chem. 259, 8037 (1984). m~T. Tennenbaum, A. K. Weiner, A. J. B61anger, A. B. Glick, H. Hennings, and S. H. Yuspa, Cancer Res. 53, 4803 (1993). ~o~F. Larcher, C. Bauluz, M. Diaz-Guerra, M. Quintanilla, C. J. Conti, C. Ballestin, and J. L. Jorcano, Mol. Carcinog. 6, 112 (1992). m2 A. B. Glick, A. B. Kulkarni, T. Tennenbaum, H. Hennings, K. C. Flanders, M. O'Reilly, M. B. Sporn, S. Karlsson, and S. H. Yuspa, Proc. Natl. Acad. Sci. U.S.A. 90, 6076 (1993). ~J3 I. M. Leigh, P. E. Purkis, A. Markey, P. Collins, S. Neill, C. Proby, M. Glover, and E. B. Lane, in "Skin Carcinogenesis in Man and in Experimental Models" (E. Hecker, E. G. Jung, F. Marks, and W. Tilgen, eds.), pp. 179-191. Springer-Verlag, New York, 1993.

20

CELLS

[2l

chloroform: 10% v/v acetic acid) or ethanol fixation (70%) is also compatible with keratin immunostaining. Expression of the hair follicle-associated enzyme y-glutamyl transpeptidase also occurs in squamous carcinomas, and this can be detected with a histochemical stain. TM It is also useful to analyze cell proliferation in the tumors by immunohistochemical detection of bromodeoxyuridine (BrdU) incorporated into DNA. 1°5 Changes in number and the distribution of labeled nuclei are generally associated with premalignant progression and malignant conversion. 1°°'1°2For BrdU staining, tumor-bearing animals are injected 1 hr before sacrifice with 250/xg/g of BrdU in 0.9% saline. Tissues fixed in either 70% ethanol or frozen in OCT can be used. Anti-BrdU antibodies are commercially available and suppliers provide the methods for use (Becton-Dickinson, San Jose, CA, Cat. No. 7580, and Zymed, San Francisco, CA, Cat. No. 93-3943).

Acknowledgment The authors thank Margaret Taylor for secretarial assistance.

104 C. M. Aldaz, C. J. Conti, F. Larcher, D. Trono, D. R. Roop, J. Chesner, T. Whitehead, and T. J. Slaga, Cancer Res. 48, 3253 (1988). 105 H. S. Huitfeldt, A. H e y d e n , O. P. F. Clausen, E. V. Thrane, D. Roop, and S. H. Yuspa, Carcinogenesis (London) 12, 2063 (1991).

[2] G e n e r a t i o n and Neuroblasts

and Culturing of Precursor Cells from Embryonic and Adult Central Nervous System

By JASODHARA RAY, HEATHER K. RAYMON,and FRED H. GAGE Introduction The mechanisms for generation of the diversified cell types of the central nervous system (CNS) from the homogeneous population of neuroepithelial cells are not well understood. Proliferating cells can be found in different brain regions at specified times during development. In general, it is thought that this developmental wave of neurogenesis ends with a departure from the cell cycle and subsequent cellular differentiation. Observations have shown that the immature CNS is not the only site for cellular proliferation METHODS IN ENZYMOLOGY,VOL. 254

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