Human cell transformation in the study of sunlight-induced cancers in the skin of man

Mutation Research, 199 (1988) 425-436 425 Elsevier

MTR 02012

Human cell transformation in the study of sunlight-induced cancers in the skin of man

Betsy M. Sutherland, Abbie G. Freeman* and Paula V. Bennett Biology Department, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.)

(Accepted 23 September 1987)

Keywords: Human cells; Skin cancer; Ultraviolet fight; DNA transfection; Anchorage-independent growth.

Summary

Human cell transformation provides a powerful approach to understanding - at the cellular and molecular levels - induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet a n d / o r near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of N IH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems.

Induction of most skin cancers in man is asso- ciated with sunlight exposure of the affected skin area. Since ultraviolet light (UV) is the major cell-damaging component in sunlight, study of transformation of human cells by UV should provide important insights into solar oncogenesis in m a n .

Correspondence: Dr. B.M. Sutherland, Biology Department, Brookhaven National Laboratory, Upton, NY 11973 (U.S.A.). * Present address: Biomedical Research Division, Lovelace

Medical Foundation, 2425 Ridgecrest SE, Albuquerque, NM 87108 (U.S.A.).

In the 1970s we developed a human cell transformation system for studying human skin cancer (Sutherland, 1978; Sutherland et al., 1980). Why a human cell system? Although several excellent transformation systems for rodent cells had been developed which gave important information on the oncogenic process, skin cancers in rodents are generally of different cellular origin than those in man. Furthermore, for reasons both of reproduci- bility and of convenience, many in vitro rodent cell transformation systems were based on immortal cell lines, quite different from the population of cells in man.

0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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In the induction of skin cancer by sunlight in man, it is likely that the initial events are photo- chemical, that is, induction of photoproducts in critical cellular molecules such as DNA. Early biochemical events probably include employment of the cellular arsenal of repair enzymes to restore the altered molecules to functional status. It seemed unlikely, however, that we could isolate, grow and characterize cells 'arrested' at either of these stages. However, since development of cancers seems to proceed by a multistage process, we thought we might succeed in obtaining from normal human cells - by UV exposure and ap- propriate selection - human cells with some of the properties of skin cancer cells. Production, characterization and determination of the relationship of such cells to the actual intermediates in development of human cancers should provide insight both into the oncogenic process in man and into the relation of the in vitro model system to human tissues in situ.

We have taken 2 approaches to understanding human skin cancer by the use of a human cell transformation system: first, production of a system for direct UV transformation of human cells (Sutherland et al., 1980), and second, development of a DNA transfection system to study transforming genes in human cancers by their action in human cells (Sutherland and Bennett, 1984). We shall discuss each approach in turn, describing our experimental approaches and results, as well as practical points and interpretations of results.

UV transformation of human cells

Design of the system In designing our human cell UV-transforma-

tion system, we tried to use conditions as similar to the normal human situation as possible, within the constraints of the requirements for maintaining cells in culture. First, many human skin cancers occur on areas subjected to repeated sunlight exposure; our laboratory counterpart of this situation is the delivery of the UV exposure in 3 equal treatments at 24-h intervals, allowing cell growth in complete medium between exposures. Second, there are 2 biological parallels suggesting a period of growth after UV and before plating: first, in man the long latent period before the appearance

of detectably abnormal cells suggests that growth or extended cell metabolism favors production of transformed cells; second, in bacterial cell muta- genesis, a period of growth under non-selective conditions for expression of the altered gene is allowed before requiring growth under selective conditions demanding use of the new gene product. We thus allowed the cells several days' growth under non-selective conditions (in plastic culture dishes in complete medium) after UV irradiation before requiring growth selective conditions. Fi- nally, we wanted a selection system which would detect human cells with altered growth properties, while not necessarily requiring complete transformation to oncogenicity (which might well require multiple steps in vitro as it does in man). We thus selected the acquisition of the ability to grow in soft agar, so called 'anchorage independence', as all human tumor cells we tested showed this prop- erty, and Shin et al. (1975) had shown good corre- lation of anchorage independence and tumorigenicity in rodent cells. We made one concession to practical considerations in our choice of human cells type: although fibroblasts are not usual pro- genitors of human skin cancers, their excellent growth and amenity to manipulation recom- mended their selection.

Experimental procedure Our system has been described in detail

(Sutherland et al., 1980; Sutherland and Bennett, 1985), and we shall summarize it only briefly here. Primary or low-passage human foreskin fibroblasts are plated in culture dishes in Dulbecco's modified Eagle's medium prepared in our laboratory (Sutherland and Oliver, 1976); after 24 h growth, the medium is removed, the cells rinsed with phosphate-buffered saline (PBS), and ex- posed to one-third of the desired total exposure of narrow- or broad-band UV radiation. After irradiation, the cells are fed with fresh complete medium, and returned to a 37°C CO 2 incubator. On the next 2 days the cells are irradiated as before, and then allowed to grow for 5 additional days. The cells are treated with trypsin, resuspended in fresh medium and counted in the presence of a vital dye to determine the concentration of viable cells; 105 cells are plated in 3 ml of 0.275% agar over an 8-ml 0.4% agar base. Each dish is scored im-

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Fig. 1. A. Human cells in soft agar immediately after plating. B. Clumps of human cells in soft agar immediately after plating.

Fig. 2. A. Untreated human cells 2 weeks after plating in soft agar. B. A human cell colony 2 weeks after plating in soft agar. Bar = 0.2 ram.

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mediately for cell clumps which might later be mistaken for anchorage-independent colonies. The cells are allowed to grow for 2 weeks, with periodic addition of complete medium. The number of anchorage-independent colonies is determined by counting all colonies in positions not previously marked as containing cells clumps. Colonies from about 0.1 mm to several mm in size are counted, depending on the cell type and history. Fig. 1A shows cells immediately after plating and Fig. 1B shows cell clumps which should be scored (immediately after agar plating). Fig. 2A shows untreated cells 2 weeks after plating and Fig. 2B shows an anchorage-independent colony.

We find that with careful attention to a few areas, human cells can be easily and reproducibly transformed by UV light.

General approach Success in the cell transformation system hinges

on the optimization of growth conditions for the particular cell type, choice of stringent selection conditions and, once the system is established, testing each new component (e.g., lot of serum) for its ability to support good growth of transformed cells while discriminating against growth of normal cells.

Cells We establish primary cultures from human

foreskins obtained from local hospitals immediately after circumcision. We find that good cell growth is absolutely essential to successful cell transformation; in our medium, such cells have good morphology and a doubling time of ap- proximately 22-24 h. It is also essential that the cells be free of all contamination, including bacteria, fungi and mycoplasma; the former over- grow the human cells in soft agar, while mycoplasma contamination can induce anchorage-independent growth (see Macpherson, 1973). Our cell stocks are grown in the absence of all antibiotics, and tested regularly by the method of Chen (1976) for mycoplasma contamination.

Irradiation We have successfully used broad-band UVB

(290-320 nm) or UVA (320-400 nm), as well as narrow-band UV (254 nm from a low-pressure

mercury arc, or radiation from a large, high-inten- sity monochromator) to transform human cells to anchorage-independent growth (Sutherland et al., 1978, 1980, 1981; Sutherland, 1982). Although the division of the total exposure into 3 treatments is sometimes not convenient, especially in large experiments such as action spectra, the increased cell survival and frequency of AI colonies - in addition to simulation of multiple exposures of man to sunlight - easily compensate for the additional procedures.

Growth after irradiation Transformation frequencies of UV-irradiated

human cells are greatly increased by allowing growth in plastic culture dishes in complete medium. This growth period was chosen to provide non-selective conditions for expression of genes altered by UV or by cellular response to UV damage. In addition, these conditions may also favor the growth of some subpopulations of cells, increasing their frequency in the cells plated in agar. During the growth period it is essential to maintain the cells under carefully controlled conditions of temperature and humidity; we find that Billups-Rothenberg chambers (Billups-Rothen- berg, Inc., Del Mar, CA; plastic gas-tight chambers) provide constant conditions and an inexpen- sive way of extending CO 2 incubator space. The frequent opening of the CO 2 incubators and re- suiting fluctuation in air temperature and CO 2 level may account for the greater success of experiments maintained in plastic chambers in our laboratory.

Plating and growth in soft agar In our early experiments we followed Macpher-

son's method for soft-agar growth of cells (Mac- pherson, 1976). This method has been uniformly successful, and we have made no major changes in the original method (see Sutherland and Bennett, 1985). Plates are poured, allowed to set and used within about 2 h; attempts to store them result in formation of a dry crust or weeping of the agar, with resulting poor adhesion of the upper agar layer. Although we have explored methyl cellulose and agarose, the former was subject to inhomo- geneity and the former provided less stringent selection than did agar (see below). Since agar is a

natural product, we test each new lot for adequate discrimination against normal cells, while allowing growth of transformed cells. We use Difco Bacto- Agar, and have not found great differences among lots over about 13 years. Since this step provides the major selection process in the transformation system, it is essential that the selection of medium, procedures for trypsin treatment, agar conditions, incubation conditions, etc. be carefully optimized for each cell type. We find that scoring each dish - although time-consuming - permits isolation of colonies from the experiment without concern that time may be wasted in characterizing colonies which actually arose from cell clumps. The cells should be sufficiently well separated during the trypsin treatment so that clumps are very rare (at most 1-2 per plate).

Maintenance of soft-agar plates Development of good transformed colonies

without growth of normal cells requires the maintenance of the soft agar at the correct moisture level by periodic feeding of the plates with small amounts (0.5 ml) of complete medium. The frequency of feeding must be adjusted according to the humidity in the incubator. The agar should remain moist but not awash, and the pH indicator should maintain a color indicating proper pH. Unless medium is added to the plates periodically, cell growth will produce sufficient quantities of acid to retard further growth of the colonies. Just before counting, it is sometimes advisable to let the plates dry slightly to facilitate counting.

Expansion of colonies from soft agar Characterization of the anchorage-independent

cells from soft-agar colonies requires their expansion to larger cell numbers than obtainable by continued growth of the original colony in the agar. After many trials of procedures for picking and growing such colonies, we found the following to give good results: T25 cell culture flasks are prepared by the addition of 1 ml of complete medium containing serum which has been tested for good ability to support the growth of cells at low density (for example, in colony-forming assays). Using a 25 × dissecting microscope, the ex- perimenter picks each colony individually from soft agar using a fine-tipped pipette; each colony

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is placed individually in a T25 flask and pipetted vigorously with a 1-ml disposable glass pipette to free the colony from the agar plug. The flasks are gassed with 5% CO 2, placed at a slight angle in the incubator, and left undisturbed for at least 1 day. They are checked, a mark is made on the top of the flask for easy later identification of the attached colony and they are allowed to grow undisturbed until the colony has grown to considerable size. They are then resuspended by trypsin treatment and replated into a T25 flask.

The many manipulations, platings, etc. in the multiple irradiations and agaring allow oportuni- ties for chance contamination of the cells. It is essential, therefore, that the resulting cells be checked for contamination, especially with mycoplasma. We also check for characteristic human isozymes (lactate dehydrogenase and glucose- 6-phosphate dehydrogenase) to confirm their iden- tity as human, non-HeLa cells (see Shannon and Macy, 1973; Gartler and Farber, 1973).

Results

Production of transformants UV irradiation of human cells produces trans-

formants which can grow to very large colonies in soft agar under ideal conditions. Fig. 2B shows a typical colony about 2 weeks after plating in soft agar. By contrast, unirradiated cells generally do not divide in the agar; Fig. 2A shows a typical field of cells from such a plate of control cells 2 weeks after plating in soft agar. We found that early-passage (cell doubling) cells yielded higher transformation frequencies (Sutherland et al., 1980), and have generally used cells of less than passage 7-10 for our studies.

Molecular target for UV transformation Since UV transforms human cells to

anchorage-independent growth, it was important to determine which cellular component was the critical target for transformation. One powerful tool for determining which cellular moiety ab- sorbed the transforming radiation is measurement of an action spectrum, that is, determination of the relative effectiveness of different UV wavelengths in transformation. The irradiations must be carried out under conditions in which reciprocity of time and fluence hold, that is, the biological

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Fig. 3. Production of transformants by 265-nm radiation at a

constant photon flux for 15,30 and 45 set (0) or for a constant

exposure time at varying photon flues (0). The points are the

averages of 10 replicate plates, and the bars show the standard

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(Sutherland et al., 1981) for killing of human cells (Kantor et

al., 1980) (o), and for pyrimidine dimer formation in V79 cells

(Rothman and Setlow, 1978) (A). The curve shown by the solid

circles represents the reciprocals of photon fluences required to produce 75 transformants per IO6 viable cells, and that shown

by the solid triangles those for the production of 150/106 cells.

effect depends on only the number of photons delivered, not the time in which they were delivered (see Jagger, 1967). The similarity of the curves in Fig. 3 shows that reciprocity held for the times and exposure rates used in our experiments (Sutherland et al., 1981). We measured transformation by the wavelengths 248, 265, 270, 280, 289 and 297 nm (the principal mercury lines). Fig. 4 shows that the action spectra obtained for transformation of human cells (filled symbols; Suther- land et al., 1981) are similar to other spectra for biological/biochemical events mediated through a

nucleic acid chromophore: human cell killing (open circles), and pyrimidine dimer formation in rodent cells (open triangles). (It should be noted that it is not usually possible, on the sole basis of action spectra, to distinguish between events mediated through DNA damage and those mediated through RNA damage, since the absorption spectra of the 2 nucleic acids are quite similar.)

Photoreactivating enzymes, as far as is known, act only on pyrimidine dimers in DNA (Setlow et al, 1965; Sutherland et al., 1985), monomerizing them in a light-dependent reaction. Expression of photoreactivating enzyme in human cells seems to depend critically on the medium in which the cells are grown (Sutherland and Oliver, 1976); human cells grown in such medium can reverse UV-induced dimers in their DNA, and exposure of UV-irradiated cells to photoreactivating light results in partial abrogation of the transforming effect of UV (Sutherland, 1978). These results suggest that pyrimidine dimers play a role in the UV transformation of human cells, but also support the involvement of other photoproducts in the transforming process.

We have used human cell transformation to probe the potentially detrimental effects of agents applied to human skin to prevent sunlight-induced damage (Sutherland, 1982). A principal UVB-ab- sorbing component in commercial sunscreening lotions is para-aminobenzoic acid (PABA), shown by Hodges et al. (1977) to sensitize pyrimidine dimer formation and mutation in bacterial cells. Indeed, in the presence of UVB radiation, PABA sensitizes the production of dimers in the DNA of human cells in culture; it also greatly increases the yield of anchorage-independent colonies induced by ultraviolet radiation (Sutherland, 1982).

Characterization of anchorage-independent cells Cells grown from soft-agar colonies frequently

grow rapidly to high density, and can attain 4-5 times the population density of untreated fibroblasts. Different colonies show variable morphology. We found many cultures with extended life spans, some reaching as many as 90-100 population doublings, versus about 50-55 for the untreated cells (Sutherland et al., 1980). No immortal cell lines have been found. We have tested a limited number of cultures for tumorigenicity in the immune-deficient mouse; no tumorigenic tines were found (although 1 mouse died of unknown causes).

When replated in soft agar the individual cultures (derived from different agar colonies) show quite different frequencies of anchorage-independent growth. Some grow at levels as high as 1-3%, while others show only about one-tenth of that value. These numbers should be compared with about 10% for fully transformed tumorigenic human cells and about 0.0001-0.001% for untreated cells in our agar conditions.

Untreated cells do occasionally form colonies in soft agar, and it would be intriguing to know if they are true spontaneous transformants to anchorage independence or arose from clumps missed during scoring. However, we have had poor results in coaxing such colonies to grow, and this characterization is not yet possible. In addition to these factors which can affect growth in soft agar, the incubator conditions must be correct for optimal colony formation. Kakunaga (1980) has discussed the striking effect of different incubators in his laboratory on the plating efficiency of one mammalian cell type in one batch of medium. We find that is is essential to optimize the CO 2 level and maintain it at that level, as well as maintaining constant incubator temperature. We measure the CO 2 level using a Fyrite gauge (Bacharach Instrument Co., Pittsburgh, PA), and open the incubator as tittle as possible; we use a water-jacketed incubator for temperature maintenance with minimum fluctuation.

Discussion

At the center of our studies is the trait selected for by growth in soft agar, so called 'anchorage independence'. Human tumor cells grow in soft

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agar (in our system, with a plating efficiency of about 10%); under proper conditions, normal cells do not. This should not be interpreted to mean that normal cells are unable to grow in all semi- solid media; agar contains several inhibitors of growth of normal cells, such as acidic and sulfated polysaccharides. Removal (e.g., production of agarose from agar) or complexing of these molecules (for example, with DEAE dextran) permits the growth of some cells unable to grow in untreated agar (for a full discussion, see Macpher- son, 1973). Macpherson also enumerates several factors which promote growth in soft agar: mycoplasma infection, addition of a layer of feeder cells, insulin, conditioned medium, high serum concentrations, etc. Thus it is essential to use selection conditions for each cell strain which do not permit growth of normal cells, but allow growth of transformed cells. (For setting up the system, normal fibroblasts can serve as negative control cells, while human tumor cells or virally transformed human cells can be used as positive controls.)

Agar growth provides a convenient screen for cellular transformation. We find that 105 cells can be plated in a 3-ml top layer over an 8-ml base in a 60-mm plate; at high cell densities (106 per plate), excessive acid production by the growing cells inhibits growth and results in plates with multitudes of very small colonies, while low cell densities (103-104 per plate) give low frequencies of growth of even fully tumorigenic human cells.

What change(s) in the cells correspond to the acquisition of the ability to grow in soft agar? Since growth in soft agar requires not only the ability to grow without anchorage, but also the capacity to grow in the presence of inhibitors of normal cell growth, there are probably several pathways by which the ability to grow in soft agar might be acquired. The most likely candidates are production of increased quantities or more highly active growth factors, alterations in cellular exte- rior or interior membranes, or changes in structural proteins, or those involved in cell division. The elucidation of which, or how many, of these are actually involved in acquisition of soft-agar growth ability will be an important milestone in our understanding of this in vitro marker and its relationship to oncogenesis in man.

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Analysis of DNA alterations in sunlight-induced human skin cancers

The direct UV-transformation system provides a useful biological system for assaying altered growth potential of human cells induced by UV. It can reveal important responses to sunlight, and allows the acquisition of biological data pertinent to sunlight effects on man. What is the relationship of the molecular and cellular changes in the in vitro transformation of human cells by UV to those in human cells damaged by sunlight which eventually form cancers in the skin?

Analysis of this relationship requires knowledge of the critical alterations in DNAs of human skin cancers, and comparison with those produced in the in vitro system. Spectacular advances in understanding the molecular origins of some human cancers have been achieved by transformation of NIH 3T3 cells by transfection with DNAs from the cancers or from cell lines derived from these cancers (for reviews, see Cooper, 1982; Bishop, 1983). NIH 3T3s are immortal murine cells and were selected as transfection recipients because of their ability to take up exogenous DNA and to form easily recognizable loci (Shih et al., 1979).

Unfortunately, however, only 30% of all human cancers yield DNAs effective in transformation of 3T3 cells (see Cooper, 1982); among those which do not are most human melanomas (Albino et al., 1984) as well as basal-cell and squamous-cell carcinomas. Since the molecular alterations in DNA in human cancers are clearly recognized by human cells, it occurred to us that use of human cells as transfection recipients should allow analysis of oncogene alterations undetected in the 3T3 system -- perhaps including those superfluous to the conversion to oncogenicity of the immortal 3T3 cells. In addition, rodent and human cells may simply differ in the genes involved in the early steps of acquisition of oncogenicity (Kakunaga, 1980, 1981).

Development of the human cell DNA transfection system

A major experimental problem impeded these studies: human cells do not take up exogenous DNA well. This problem was solved by use of a polyethylene glycol (PEG) permeabilization system for mammalian cells previously developed in

our laboratory for the insertion of exogenous enzymes into rodent cells (Sutherland and Hausrath, 1979). R. Pollack (personal communication) had used our method for inserting entire virions into mouse L cells, suggesting that PEG made the cell membrane quite permeable indeed.

A second problem was choosing a selection system which would allow recognition of altered genes from DNAs of cancers, while giving low backgrounds for untreated cells or cells treated with DNAs from normal cells. Fortunately, the PEG permeabilization system is compatible with assessment of altered cellular metabolism by growth in soft agar; that is, PEG treatment neither permanently injures the cells nor do the membrane alterations produced by it permit soft-agar growth of normal cells (Sutherland and Bennett, 1984). Agar growth and focus formation are com- plementary assays; it seems likely that the ability to grow in soft agar represents an earlier step in transformation of human cells than does focus formation, the opposite of the situation in rodent cells.

Experimental procedures DNA preparation and insertion. DNA purifi-

cation has been described in detail (Sutherland and Bennett, 1985), and will be described only briefly here: high-molecular-length genomic DNAs are prepared by treating cells with sodium dodecyl sulfate and proteinase K, followed by phenol ex- traction and sec-butanol treatment. The DNA is dialyzed, treated with RNAses A and T1, reex- tracted as before and dialyzed until the A260 is ~< 0. The DNAs are characterized by absorption spectra (A260/A280 should be =-2) and by molecular length (electrophoresis on 0.4% agarose should indicate a molecular length of 50-100 kb).

Cells. Primary or low-passage fibroblasts from human foreskin cultures initiated in our laboratory are used as standard transfection recipients. Cell stocks are grown in Dulbecco's modified Ea- gle's medium without antibiotics prepared in our laboratory (Sutherland and 0liver, 1976), and are checked regularly for mycoplasma contamination by the method of Chen (1976). We find that good cell growth is a prerequisite to successful cell transfection and subsequent transformation.

Transfection. Different cell types vary mark- edly in their ability to take up DNA, and in reaction to PEG. The procedure we describe below works well for V79 cells (for insertion of exogenous proteins as well as DNA), and for human foreskin fibroblasts (Sutherland and Ben- nett, 1984, 1985); the use of other cell types requires determination of the opt imum conditions for D N A uptake and good cell survival. Optimiza- tion of conditions for each cell type is probably the most time-consuming and exacting part of the transfection procedure; once these conditions have been determined, the transfection experiments proceed easily and reproducibly. If the PEG procedure is being optimized for a new cell type, the work of Clark et al. (1978) will be of great help in suggesting pertinent variables.

Cells to be transfected are plated in 60-mm culture dishes at a density of 103 per dish, and allowed to grow overnight. About 30 min before adding to the cells, the DNA-CaC12 solutions are prepared by mixing the D N A into Hepes-buffered saline (Sutherland and Bennett, 1984) and adding CaC12 to 0.125 M final concentration, inverting to mix and allowing the tubes to stand without being disturbed for 20-30 min. Meanwhile, the medium is removed from the cells, they are rinsed with PBS and treated for 2 rain with a 37 ° C solution of 1 part PEG 6000 to 2 parts medium (Suther- land and Hausrath, 1979). (We use PEG from Fisher Scientific, now renamed Carbowax PEG 8000, without further purification.) The PEG solution is removed, and the cells rinsed thoroughly with PBS. Complete removal of the PEG is essential for good cell survival; traces of PEG can be detected as an observable film on the surface of the PBS.

The D N A solution is mixed with complete medium (0.5 ml D N A solution containing the required quantity of D N A is mixed with 3.5 ml medium) and applied to the cells; after 24 h incubation in a Bil lups-Rothenberg chamber, the D N A - m e d i u m mixture is removed, the cells rinsed with PBS, complete medium is then placed over the cells and they are allowed to grow undisturbed for 5 days. The 24-h incubation period for uptake of D N A by the human foreskin fibroblasts is based on experiments in which the frequency of anchorage-independent colonies was determined

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for cells allowed to take up MOLT-4 D N A for differing times; for the human foreskin fibroblasts, 24 h permitted good uptake without excessive toxicity.

Selection The transfected cells are now subjected to selec-

tion according to the markers inserted: soft-agar growth for detection of transforming sequences, antibiotic resistance for determination of uptake and expression of drug resistance markers, etc. Our procedure for soft-agar plating and growth is that used in direct UV transformation of human cells and detailed descriptions of the experimental procedure are found in Sutherland et al. (1980) and Sutherland and Bennett (1984, 1985).

Results Human cells can be transformed to anchorage-

independent growth by transfection with a wide variety of human tumors, including melanomas (Sutherland and Bennett, 1984; Sutherland et al., 1985). Table 1 shows typical results from such a transfection. Control cells treated with all components of the transfection system except D N A show very low frequencies of growth. Furthermore, fibroblasts transfected with DNAs from normal mammalian cells show agar growth frequencies at only background levels. One important point

TABLE 1

TRANSFORMATION OF HUMAN CELLS BY DNA TRANSFECTION

DNA donor Colorties/105 cells

GM 1500 (multiple myeloma) 85 + 4.2 CRL 1424 (malignant melanoma) 96.6 + 6.9 T24 (human bladder carcinoma) 42 + 2.3 pHuBlym 320 + 24.0 pT24-C3 523 + 17.7 Normal human B cells 6.6 + 2.7

PBS only 2.3 + 0.9 No DNA (+PBS, PEG, CaCI2) 1.8+ 0.6

Neonatal human foreskin fibroblasts were rinsed with PBS, permeabilized with PEG, and transfected with DNAs from human cancer cells or normal human cells (data above the line of white). Controls (below the line of white) were treated with PBS only, or with all components of the transfection system except DNA. Data are the averages of 6-10 replicate plates and are given + the standard error of the mean; all numbers are transfectants per #g of transfecting DNA.

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should be made about transfection frequencies: the absolute transformation frequencies vary con- siderably from experiment to experiment, although the relative values are similar. We generally classify DNAs into broad classes of poor, fair or good transformers, and regard the absolute numbers obtained in any one experiment only as a guide for placing a DNA in such broad classifica- tions.

Are human cells transformed by the same altered genes as NIH 3T3s? Transfection of human cells with genomic DNAs or cloned oncogenes which are highly efficient in 3T3 transformation indicates that human cells do recognize altered genes such as H-ras. DNA from the bladder carcinoma T24 is an efficient transformer of human cells. The cloned H-ras and B-lym oncogenes, however, show a low efficiency (on a per gene copy basis) in transforming human cells, suggesting that DNA configurations or flanking sequences may be important in uptake, survival, integration or expression of DNA in human cells.

We examined the effect of restriction upon the transfecting activity of GM 1500 (multiple myeloma) DNA in 3T3 versus human cells (with the kind collaboration of G. Cooper and D. Bec- ker, Dana-Farber) (Sutherland et al., 1985). Treat- ment of 1500 DNA with EcoRI, HindlII, BAMHI, XhoI or PvulI did not affect the transforming activity of 1500 DNA into either human cells or 3T3s; DNase-I treatment abolished activity in either cell type. However, SacI reduced transforming activity in 3T3s, but not in human cells. There are several possible explanations for such a result: first, human ceils might be recogniz- ing different transforming genes in 1500 DNA; second, there might be more than 1 gene in 1500 DNA able to transform human cells (and thus restriction-enzyme cleavage might not inactivate all of the transforming loci); third, SacI might remove sequences essential for integration or expression in rodent cells but not in human cells. Differences in colony morphology in soft agar among human cells transformed to anchorage-independent growth by DNA from different cancer DNAs suggest that human cancers may contain more than 1 altered gene capable of transforming human cells.

Many of the DNAs tested in our initial experi-

ments were active in transformation of N IH 3T3s. We were thus intrigued to determine whether the human cell recipient could be transformed even by DNAs which did not give a detectable effect in the 3T3 transfection system.

We first tested D N A from GM 1312, a multiple myeloma cell line shown by Lane et al. (1982) to be ineffective in transformation of N IH 3T3 cells. This DNA transformed human fibroblasts to anchorage-independent growth, although at a lower frequency than that of the multiple myeloma GM 1500 (which also transforms 3T3s) (Suther- land et al., 1985). In addition, anchorage-independent colonies resulting from primary transfectants of DNA from GM 1312 attached to plastic, grew on plastic surfaces, and plated in soft agar with about the same efficiency as did human cell transfectants of GM 1500. Bennett et al. (1986) have also shown that DNAs from melanoma cells which are ineffective in transformation of 3T3s can transform human cells to anchorage-independent growth. The transformation frequency in human cells showed no relationship to the frequency of transformation of 3T3 cells, suggesting the recognition of different oncogenes by the 2 cell types.

Since human cells can be transformed by genes not detected in 3T3 cells, it seemed possible that DNAs from normal cells might be able to mediate transformation by insertion between control ele- ments and structural genes or by other events leading to abnormal growth control. It remains a matter of some surprise to us that this does not seem to be the case: DNAs from normal mammalian sources, whether neonatal, young, adult or aged human, give transformation frequencies in the same general range as background (Bennett et al., 1986). It would be intriguing to study the occasional colony arising in soft agar in human fibroblasts transfected with DNA from normal human sources, but such colonies have proven difficult to grow to sufficient cell numbers for further characterization. This suggests a qualita- tive difference between agar colonies arising by transfection with DNAs from normal mammalian sources and those produced by transfection with DNAs from cancers or cancer cell lines.

Future directions Our scientific crystal ball suggests 3 important

areas of research critical to understanding of transforming genes in human skin cancers and their relation to in vitro transformation: first, determination of the range of oncogenes and gene products altered in human skin cancers. These may be detected by their transforming activity in human cells in vitro. Assaying transformation by different markers - for example, growth in soft agar, focus formation, formation of a non-progressing tumor in the immune-deficient mouse, and formation of a progressing tumor - should allow discrimination among and description of the genes conveying the properties of fully tumorigenic cells. The second area is the determination of molecular heterogeneity in human skin cancers. For example, is malignant melanoma really a col- lection of diseases at the molecular level? Are there only single alterations in 1 oncogene, or do more than 1 oncogenes show changes in sequence or copy number? Is there any common pattern of oncogene alteration among the various sunlight- induced skin cancers? The third area is the evalua- tion of in vitro human cell-transformation system as a model system for oncogenesis in man. Do current human cell UV-transformation systems produce cells with D N A alterations related to those found in DNAs from actual skin cancers? Do additional molecular and cellular alterations occur in cells in culture? Can better (or more complete) cell-transformation systems be found? The delineation of these areas should provide important information on molecular and cellular alterations in sunlight-induced skin cancer and the relation of in vitro UV transformation of human cells to in situ solar oncogenesis in the skin of man.

Acknowledgements

We thank Ms. Rowena Oliver, Ann Tebbut t and Anne Katz for excellent collaboration in the course of this work. This research was supported by N I H Grants CA26492 and CA23096, and by the Office of Health and Environmental Research of the U.S. Department of Energy.

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Human cell transformation in the study of sunlight-induced cancers in the skin of man