Tumorigenic conversion of immortal human skin keratinocytes (HaCaT ...

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Oncogene (1999) 18, 5638 ± 5645 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc

Tumorigenic conversion of immortal human skin keratinocytes (HaCaT) by elevated temperature P Boukamp*,1, S Popp1, K Bleuel1, E Tomakidi1, A BuÈrkle2 and NE Fusenig1 1

Division of Carcinogenesis and Dierentiation, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany; 2Division of Applied Tumor Virology, Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany

UV-radiation is a major risk factor for non-melanoma skin cancer causing speci®c mutations in the p53 tumor suppressor gene and other genetic aberrations. We here propose that elevated temperature, as found in sunburn areas, may contribute to skin carcinogenesis as well. Continuous exposure of immortal human HaCaT skin keratinocytes (possessing UV-type p53 mutations) to 408C reproducibly resulted in tumorigenic conversion and tumorigenicity was stably maintained after recultivation of the tumors. Growth at 408C was correlated with the appearance of PARP, an enzyme activated by DNA strand breaks and the level corresponded to that seen after 5 Gy g-radiation. Concomitantly, comparative genomic hybridization (CGH) analyis demonstrated that chromosomal gains and losses were present in cells maintained at 408C while largely absent at 378C. Besides individual chromosomal aberrations, all tumor-derived cells showed gain of chromosomal material on 11q with the smallest common region being 11q13.2 to q14.1. Cyclin D1, a candidate gene of that region was overexpressed in all tumor-derived cells but cyclinD1/ cdk4/cdk6 kinase activity was not increased. Thus, these data demonstrate that long-term thermal stress is a potential carcinogenic factor in this relevant skin cancer model, mediating its eect through induction of genetic instability which results in selection of tumorigenic cells characterized by gain of 11q. Keywords: transformation; heat; comparative genomic hybridization; PARP activity; chromosome 11

Introduction In human skin carcinogenesis UV-B has been identi®ed as the major carcinogenic risk factor. There is accumulating evidence that UV-B is responsible for mutational inactivation of the p53 tumor suppressor gene and that this event initiates skin carcinogenesis (for review see Brash, 1997). Skin carcinoma as well as premalignant skin lesions carry p53 mutations which are indicative for UV-B damage, namely C to T transitions in CC sites or CC to TT double base exchanges (Brash et al., 1991). A few cases of squamous cell carcinomas (SCCs) also show mutational activation of the ras gene (for review see

*Correspondence: P Boukamp Received 14 January 1999; revised 27 April 1999; accepted 27 April 1999

Ananthaswamy and Pierceall, 1992). Most of these mutations are C to T transitions in codon 12 of the Harvey or Kirsten ras gene located opposite a CC site, and also for these mutations UV-B is discussed as the causal mutagenic agent. Epidemiologic and experimental data indicate that in addition UV-A has to be considered as a risk factor which may account for at least 20% of the chromosomal aberrations found in skin carcinomas (for review see de Laat and de Gruijl, 1996). Evidence is mainly derived from animal models which showed that UV-A was able to induce benign (papillomas) and malignant tumors (SCC) (de Laat et al., 1996). However, little is known so far about the nature of the genetic damage caused by UV-A. Wave-lengths above 347 nm (UV-A: 315 ± 400 nm) do not cause DNA damage by direct absorption of photons (Peak et al., 1987), so that DNA damage is supposedly induced by indirect mechanisms, i.e. reactive oxygen species causing single and double strand breaks (Peak and Peak, 1982). As a third factor, elevated temperature, as measured in areas of sunburn, may contribute to skin carcinogenesis by generating DNA damage via the same indirect oxidative stress pathway (Cerutti et al., 1990). Recently, it was shown that rat embryo ®broblasts when cultured at 398C as compared to 378C exhibited an increased number of strand breaks and that these cells became tumorigenic when maintained at 398C for 47 days (Marczynska et al., 1980). Short-term exposure (*1 h) of cells to 4428C, although by itself not tumorigenic, acted synergistically when combined with g-radiation (for review see de Gruijl, 1996). In order to test whether elevated temperature is a cofactor for human skin carcinogenesis, we exposed immortal human skin keratinocytes to an increased, albeit still physiologically tolerable, temperature (408C) over an extended period of time. The HaCaT cells used for these studies represent an early stage of skin carcinogenesis by exhibiting UV-B type-speci®c mutations in the p53 tumor suppressor gene (Lehman et al., 1993). They are further characterized by a well de®ned genetic make-up and a largely balanced chromosomal content also during long-term culture as demonstrated by comparative genomic hybridization (CGH) (Boukamp et al., 1988, 1997). Moreover, they remained nontumorigenic over more than 300 passages when propagated under standard culture conditions at 378C. We now demonstrate that these HaCaT cells when cultured at 408C for 15 ± 20 passages reproducibly acquire a tumorigenic phenotype in nude mice. This tumorigenic progression is

Keratinocyte carcinogenesis by 408C P Boukamp et al

preceeded by changes in the CGH pro®le suggesting that elevated temperature causes genetic instability resulting in chromosomal imbalance. All cell lines established from nude mouse tumors show gain of genetic material on chromosome 11q suggesting that ampli®cation of genes on 11q is causal for tumorigenic conversion.

Results Continuous growth at 408C leads to tumorigenic conversion HaCaT cells grown at 378C up to passage 90 were transfered to 408C and maintained at this temperature further on (90T-A). After an initial phase (*2 weeks) of increased cell death and thus prolonged time to reach con¯uence all subsequent passages were performed at weekly intervals at a split ratio of 1 : 10. The tumorigenic potential was tested by s.c. injection into immunode®cient nude mice. No tumor growth was detected with cells grown at 408C for three passages (Figure 1a), whereas with cells from passage 16, two out of ®ve mice developed tumors (Figure 1b). These tumors were histologically classi®ed as locally invasive squamous cell carcinomas (SCC), i.e. malignant tumors (Figure 2a). The same set of experiments was repeated with two other HaCaT populations, i.e. another independent batch of cells from passage 90 and cells from passage 25 (25T). Again, HaCaT cells from passage 90 (90T-B cells) showed no tumor growth when grown at 408C for two passages, while passage 13 cells formed small but persistent nodules in two mice. After 21 passages at 408C, cells formed one rapidly expanding and two slowly enlarging tumors which by histology were all locally invasive SCCs, i.e. malignant tumors (data not shown). Tumorigenic conversion by growth at 408C was not restricted to late passage HaCaT cells but similarly occurred with cells from passage 25 (25T). After growth for one and 11 passages at 408C the cells were still nontumorigenic. However, after 20 passages, three out of six mice developed tumors. In contrast to those tumors obtained with later passage HaCaT cells, all three (25T) tumors were large epidermal cysts with a well dierentiated lining epithelium (Figure 2b). Because they did not show any local invasion, they were classi®ed as benign tumors.

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Elevated temperature induces DNA strand breaks In order to establish a possible mechanism by which growth at elevated temperature (38C) had resulted in tumorigenic conversion of the HaCaT cells, we searched for indications of DNA damage. The activation of poly(ADP-ribose)polymerase (PARP), a nuclear enzyme which is involved in DNA base excision repair, was used as an indicator of DNA strand break formation in living cells. In the presence of DNA strand breaks (single- or double) the enzyme gets stimulated to modify acceptor proteins by covalently attaching ADP-ribose moieties. This results

Figure 2 Histological evaluation of the tumors developed by 408C HaCaT cells (a) 90T-A cells (experiment I) formed locally invasive (arrow head) solid SCCs, i.e. malignant tumors while (b) 25T cells developed epidermal cysts with an epidermis-like lining epithelium (arrow). Insert: Higher magni®cation of the rather normal tissue architecture of the lining epithelium. Bar=100 nm

Figure 1 Tumor growth curves of HaCaT cells grown at 408C and injected into thymus aplastic nude mice. (a) HaCaT cells of passage 90 (90T-A) (experiment I) were analysed for tumor growth after 3 (p3) and (b) 16 passages (p16) at 408C, respectively. Note that after 16 passages at 408C two of the ®ve mice developed tumors. (c) Recultivated and reinjected tumor cells (90T-A/rt-1) formed tumors in all but one mouse and with a reduced latency period


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in the formation of chains of poly(ADP-ribose) which can be visualized in the nucleus with a PARP-speci®c antibody (BuÈrkle et al., 1993). As a positive control, cells were treated with g-radiation, a well-known method to induce DNA strand breaks. PARP was activated after g-radiation in a dosedependent manner with a maximum of *90% PARPpositive nuclei after 30 Gy (Figure 3a), *70% after 20 Gy, and *1% after 10 Gy (Figure 3b). After gradiation with 5 Gy still scattered single cells stained positive. No positive cells were detected in control cultures (maintained at 378C) while PARP was activated in cultures at 408C. The maximum frequency of positive cells was seen after 32 ± 36 h exposure to 408C and equivalent to the number of positive nuclei seen after g-radiation of 5 Gy (Figure 3c). At later time points, poly(ADP-ribose) formation was only rarely observed. In living cells poly(ADP-ribose) is undergoing a turnover, with half-lives ranging from about 8 h in untreated cells to less than a minute in cells exposed to high-dose alkylating treatment which induced extensive DNA breakage and consequently strong PARP activation (Alvarez-Gonzales and Althaus, 1989). Furthermore, hyperthermia (4428C) can cause a decreased turnover rate of ADP-ribose polymers (Jonsson et al., 1988a,b). Thus, to exclude that the poly(ADP-ribose) immuno¯uorescence seen at 408C was caused by an accumulation of poly(ADP-ribose) due to a decreased degradation rather than increased de novo synthesis, we treated the cells with the PARP inhibitor 3-aminobenzamide 1 and 2 h before assaying for PARP activity (BuÈrkle et al., 1993). g-Radiation treated control cells showed no PARP activation in the inhibitor-pretreated cells. Similarly, no positive cell could be detected in the 408C cultures treated with 3aminobenzamide for 1 or 2 h (Figure 3d). Thus, our data strongly suggest that the poly(ADP-ribose)

Figure 3 Immunostaining with a PARP-speci®c antibody to identify cells with DNA strand breaks. After g-radiation with (a) 30 Gy, (b) 10 Gy (c), and in a culture of HaCaT cells 32 h after transfer to 408C. (d) PARP activity is completely inhibited in cells pretreated with 3-aminobenzamide 1 h prior to assaying for PARP activity

detected in cultures at 408C was not caused by increased half life but by de novo formation triggered by induction of DNA strand breaks. Continuous growth at 408C favors accumulation of genetic aberrations To investigate whether DNA strand breaks induced by growth at 408C were of any consequence for the chromosomal balance of the HaCaT cells, the entire genome was screened for chromosomal gains and losses by comparative genomic hybridization (CGH) (Kallionemi et al., 1992). Cells which were transferred to 408C and had survived the initial phase of increased cell death had retained the chromosomal aberrations characteristic for HaCaT cells grown at 378C as shown for 90T-B cells grown at 408C for 11 passages. These are underrepresentation of chromosomes 3p, 4, and 9p due to the early loss of one respective copy and ampli®cation of 9q due to an early reduplication [i(9q)] (Figure 4a and for comparison see Boukamp et al., 1988, 1997). Similarly, underrepresentation of chromosomes 18 and 21 were already present in HaCaT cells cultured at 378C. In addition, the 408C HaCaT (90T-B) cells showed three new aberrations, i.e. gain of 6q (which was underrepresented in 378C HaCaT cells and therefore now appears in a balanced state, in blue), gain of Xq (in green), and loss of at least one copy of 20 (which was overrepresented at 378C) (Table 1). With each further analysis (at about ten passage intervals), a set of new aberrations emerged (see Table 1) and this was a consistent ®nding in all three experiments. The individual gains (+) and losses (7) are listed in Table 1. A common genetic aberration characterizes three independent tumorigenic HaCaT populations The above data showed that prolonged cultivation at 408C favored steady evolution of genetically divergent subpopulations. Furthermore, the long latency period of tumor growth and the restricted number of tumors suggested that only a minor cell fraction of the mass population had become tumorigenic. In order to determine the genetic subpopulation responsible for growth in vivo, tumors from the three individual experiments were recultivated (90T-A/rt-1, 90T-B/rt, and 25T/rt) reinvestigated for tumor formation, and analysed by CGH. After reinjection into nude mice, all recultivated tumor cells formed tumors in all (except 1) mice (Figure 1c) and with reduced latency periods. Thus, tumorigenicity was a stable trait of these cells. Similarly, the tumor phenotypes remained unchanged with being malignant for 90T-A/rt- and 90T-B/rt cells and benign for 25T/rt cells. The CGH pro®les of these recultivated tumor cells were very similar to the respective 408C mass populations clearly proving their common origin. In addition, each tumor reconstitute showed individual chromosomal gains and losses thereby demonstrating their independent development (Table 2). Remarkably, all tumor-derived cell populations also contained one common aberration, i.e. gain of 11q with the smallest common region being 11q13.2 to q14. The overall chromosomal region diered with 11p11.1 to q14.2 in 90T-A/rt- cells, 11q13 to q14.2 in 95T-B/rt cells, and 11q11 to q22 in 25T/rt cells (see Table 2 and Figure


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1

2

3

4

Figure 4 CGH analysis of 408C HaCaT cells. (a) Color scheme (left), idiogram (middle), and ¯uorescence ratio pro®le (right) for each chromosome from 90T-B cells (experiment II) grown at 408C for 11 passages. In the color scheme, the blue color represents normal DNA content, green indicates gain and red loss of chromosomal material. All aberrations marked in red and green were already preexistent in HaCaT cells grown at 378C (for comparison see Boukamp et al., 1997). In the ratio pro®les, the three vertical lines represent the balanced state (middle) of the chromosomal material, the upper (right), and lower (left) threshold, indicative of gains and losses of chromosomal material, respectively. Gray represents areas of heterochromatin which are not included in the evaluation. (b) Dapi-stained chromosomes 11 (left), FITC/TRITC ratio image (middle), and CGH pro®les (right) from the (1) 90TA, (2) 90T-A/rt-2a, (3) 90T-B/rt, and (4) 25T/rt cells, respectively demonstrate that gain of 11q is only present in the three tumorderived populations (two to four) (green areas in the chromosomes and shift to the outer right lane in the CGH ratio pro®le) while in the 90T-A cells (1) chromosome 11 is in a balanced (blue) state

4b). Gain of 11q was not seen in any of the three HaCaT populations grown at 408C prior to the injection into nude mice (see Figure 4a, Table 1) but was again prominent in cells (90T-A/rt-2a and b) recultivated from tumors induced by the recultivated tumor cells 90T-A/rt-1 (see Table 2). Cyclin D1 as a candidate oncogene Of a number of potential genes mapped to the chromosomal region of 11q13 cyclin D1 was a good candidate gene responsible for the observed tumorigenic conversion. By Western blot analysis an increased level of cyclin D1 was detected in all tumor-derived cells, all showing gain of 11q, as compared to cells grown at 408C and which had no detectable gain of 11q (Figure 5a). Cyclin D1 is, however, only active as a cell cycle regulator when complexed with cdk4 or cdk6. We, therefore, additionally performed a kinase assay in which Rb was used as a substrate (Matsushime et al., 1994). Surprizingly, no consistent dierences could be detected between the tumor-derived cells and the parental noninjected 408C populations (Figure 5b). Substantial amounts of phosphorylated Rb were seen

in all cells, demonstrating that despite increased cyclin D1 protein levels kinase activity was not altered. Discussion In addition to UV-B which induces point mutations and UV-A which is believed to cause indirect DNA damage via oxygen radicals, we here provide evidence that long-term exposure to increased temperature as encountered in skin areas exposed to excessive sunlight, i.e. in sunburn, and which are believed to be the areas of high risk for tumor development, may act as a cofactor in skin carcinogenesis. There is accumulating evidence that growth at elevated temperature may enhance cell transformation. (i) The HaCaT cells, used as a model in the present experiments, became immortal, while grown at increased temperature (Boukamp et al., 1988). (ii) Short-term heat exposure (4428C for 60 ± 90 min) had an additive eect on tumorigenic conversion when followed by g-radiation (for review see de Gruijl, 1996). (iii) Rat embryo ®broblasts became tumorigenic when cultured at 398C for 47 days (Marczynska et al., 1980). The ®rst and most important ®nding of this study is that a


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Table 1 Chromosomal imbalances in HaCaT cells grown at 408C as determined by comparative genomic hybridization 90T-A (experiment I)

90T-B (experiment II)

25T (experiment III)

CGH Karotype after 3 passages at 408C



rev ish enh: (gains) (3q, 7p, 9q, 17p11q12, 20, Xq11q13, Xq24q25)

rev ish enh: (gains) (Xq25qter),

rev ish enh: (gains) (9q),

dim: losses (3p, 4, 9p21qter, 21q, Y)

dim: (losses) (3p, 4, 9p21qter, 18p11.1p11.2, 21q, Y

dim: (losses) (1p, 3p, 4p, 4q28qter, 9p, 18, Y)

a

Additional 13 passages at 408C

Gains (+) Losses (7)

Additional



+ (8q) 7 (18p)

25 passages at 408C

+ (7p) 7 (10p) 7 (18)(p11.3)

22 passages at 408C

+1 +3 +7 + (8q) + (11)(q147q23) + (13q) +16 +17 +18 +20 + (X)(q23 ± 27)

31 passages at 408C

+ (17)(q12 ± pter) +20

28 passages at 408C

7 (8q) +(15)(q117q15) +X

Additional

a According to ISCN (1995): rev ish, reverse in situ hybridization including comparative genomic in situ hybridization; enh=enhanced ¯uorescence intensity ratio indicating an increased copy number (gain +); dim=diminished ¯uorescence intensity ratio indicating loss (7) of chromosomal material

Table 2 Chromosome aberrations characterizing the recultivated tumor cells in addition to those listed in Table 1 90T-A/rt-1

90T-A/rt-2a

90T-A/rt-2b

75

75 +(8)(p21.2 ± pter)

75 +(8)(p21.2 ± pter)

+(11)(pter7q13)

+(11)(p11.17q14.2)

+(11)(p147q21)

+(14)(q117q21)

+(14)(q137q24.1)

+(X)(p11.47qter)

719q +(X)(p11.27qter)

temperature increase of 38C was sucient to reproducibly induce tumorigenicity in these immortal though stably nontumorigenic human skin keratinocytes within 13 ± 20 passages. As a second major result we show that tumorigenic conversion occurred independent of the passage level of HaCaT cells. These data demonstrate that long-term exposure to elevated temperature can act as a cofactor in the carcinogenesis process. Although all cells became tumorigenic, HaCaT cells from dierent passage levels exhibited dierent phenotypes. While early passage cells (25T) gave rise to benign tumor cells, later passages (90T) reproducibly formed malignant tumors. Comparable results were obtained when HaCaT cells were rendered tumorigenic by introduction of the rasH oncogene (Boukamp et al., 1990, 1995) or following long-term culture under serum free conditions (Hill et al., 1991). This clearly demonstrates that long-term culture at elevated

90T-B/rt

25T/rt

7(2)(q23-qter) +5p

+(8)(q23 ± qter) 710p +(11)(p) +(11)(q117q22) +(11)(q137q14.2) +(13)(q317qter) 7(18)(q22.37qter) 7(18)(q227qter) 7Xp

Figure 5 Characterization of cyclin D1. (a) Comparison of cyclin D1 protein expression by Western blot analysis of 90T-A cells (without gain of 11q), the tumor-derived 90T-A/rt-1 and two tumors derived from reinjected 90T-A/rt-1 cells, 90T-A/rt-2a and 90T-A/rt-2b (all with gain of 11q). (b) Comparison of cyclin D1/ cdk4/cdk6 kinase activity between the nontumorigenic and tumorderived cells from the three experiments. Cell extracts were immunoprecipitated with a cyclin D1 antibody and the immune complexes were assayed for pRb kinase activity


temperature has a similar potential as e.g. the rasH oncogene in that it can induce genetic changes required for tumorigenic conversion. But the tumor phenotype, i.e. the stage of transformation, seems to be determined by the preexisting genetic make-up of the cell. This is in good agreement with the ®nding that also benign and malignant skin tumors (keratoacanthomas and squamous cell carcinomas) share some common genetic alterations and that e.g. rasH oncogene is among those common aberrations (Corominas et al., 1989). As a potential mechanism by which elevated temperature is causing tumorigenic conversion we propose that 408C is able to induce DNA strand breaks which eventually result in chromosomal aberrations. This hypothesis is based on two ®ndings: First, PARP is activated when HaCaT cells are grown at 408C while absent in cells grown at 378C. PARP is an eukaryotic enzyme that speci®cally recognizes DNA strand breaks. Binding to strand breaks triggers catalytic activity of PARP resulting in the covalent modi®cation of nuclear proteins, including PARP itself, by poly ADP-ribosylation. While the steadystate levels of poly(ADP-ribose) in intact cells are extremely low under normal conditions (378C), the biopolymer was rapidly detected by immunofluorescence in cells cultured at 408C and the number of positive cells suggests that the maximal induction of PARP at 408C is comparable to that of about 5 Gy gradiation. This is a dose which is well compatible with cell survival as shown previously when HaCaT cells were still able to form colonies after irradiation with doses 510 Gy (Mendonca et al., 1991). Furthermore, the experiments using the ADP-ribosylation inhibitor 3-aminobenzamide further demonstrated that the poly(ADP-ribose) immunostaining seen at 408C was not due to its altered stabilization by hyperthermia. Instead, the data strongly suggest de novo synthesis as a consequence of PARP activation by increased temperature-induced DNA strand breaks. In agreement and most likely as a consequence of DNA strand breaks, new chromosomal gains and losses were detected by comparative genomic hybridization (CGH) at each passage analysed. This was in marked contrast to cell growth at 378C where the CGH pro®les remained largely unchanged during long-term passaging (Boukamp et al., 1997). Moreover, by characterizing the aberration pattern at dierent stages of cultivation and after in vivo passage we were able to identify one aberration, i.e. gain of 11q, as a marker for all tumorigenic cell populations. Gain of 11q is seen in a number of tumors (Bockmuhl et al., 1996; Brinkschmidt et al., 1997; Mahlamaki et al., 1997; Tirkkonen et al., 1998) but was so far not described for skin carcinomas. Own preliminary data indicate a similar gain of 11q (including the area of q13) in skin SCC cell lines and primary tumors (SCCs and benign keratoacanthomas) (Popp and Boukamp, in preparation). Collectively, this may suggest that gain of 11q, because being present in both benign and malignant tumors, is relevant for early skin carcinoma development. One of the genes mapped to 11q13 and likely to be involved in altered growth control is cyclin D1, a regulator for cell-cycle progression through G1 (for review see Sherr, 1996). The level of cyclin D1 was increased in all tumor cell populations. Ampli®cations of 11q13 and/or overexpression of cyclin D1 have been

described for an increasing number of tumors (Nakagawa et al., 1995; Naitoh et al., 1995; Sheyn et al., 1997) and recently, overexpression of cyclin D1 was also suggested as a marker for skin carcinomas (Inohara et al., 1996). Because cyclin D1 is only operative when complexed with its cyclin-dependent kinase (cdk4/cdk6) we additionally determined kinase activity. To our surprise, kinase activity remained largely unchanged. Thus, it remains to be seen whether overexpression of cyclin D1 has nevertheless a functional consequence on cell cycle control and was causal for tumorigenic conversion of HaCaT cells. In conclusion, our studies show that long-term exposure to a moderately elevated temperature (408C) is eective in promoting tumorigenic progression of immortal human HaCaT skin keratinocytes which represent an early stage in skin carcinogenesis. Exposure to 408C obviously induced DNA strand breaks and these were followed by and thus are likely to be causal for chromosomal gains and losses. During continuous in vitro propagation at 408C genetically altered subpopulations emerged. One of those, characterized by 11q ampli®cation, had obviously acquired a selective in vivo growth advantage. Because this aberration was present in all tumor-derived cells, it is tempting to speculate that gain of 11q may be speci®c for temperature-stress induced tumorigenic conversion. Material and methods Cells and culture conditions HaCaT cells were routinely cultured as described (Boukamp et al., 1988) and tumor formation was analysed as previously described (Bleuel et al., 1999). To recultivate cells from nude mouse tumors, these were excised, quickly rinsed in 70% ethanol, thoroughly washed in MEM culture medium and minced. Individual pieces were transfered to culture dishes, air dried for a minute, and carefully immersed in MEM culture medium containing 10% FCS. Contaminating ®broblasts were eliminated by short-term exposure to 0.05% trypsin. Epithelial islands were trypsinized and passaged as described (Boukamp et al., 1988). Genetic analysis by comparative genomic hybridization (CGH) DNA was isolated from HaCaT cells at dierent passages at 408C and CGH analysis, image acquisition, and processing were performed as described previously (Boukamp et al., 1995). Average ratio pro®les of ¯uorescein-labeled HaCaT DNA and texas-red-labeled normal DNA were calculated from at least ten metaphase spreads and represented schematically. The central line in the pro®les represents the most frequently measured ¯uorescence ratio for each metaphase spread, corresponding to a balanced state of chromosomal material. The right and left vertical lines de®ne threshold values for over- and underrepresentation of chromosomal material (du Manoir et al., 1995). Immuno¯uorescence analysis of poly(ADP)-ribose production in intact cells Cells were plated onto glass coverslips and grown up to about 50% con¯uency. After treatment with g-radiation (30, 20, 10, and 5 Gy) cells were ®xed in 10% trichloroacetic acid on ice for 10 min. Temperature eects were measured by incubating cells growing on coverslips at 378C and 408C, respectively, for 2 h up to several days prior to ®xation. Thereafter, samples were dehydrated in graded ethanol and

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air dried. Immunostaining procedure was performed as described previously (BuÈrkle et al., 1993) using a monoclonal antibody (10 H) against poly(ADP-ribose) at a concentration of about 5 mg/ml in PBS/1% BSA. The speci®city of the antibody was determined as described (Kawamitsu et al., 1984). The second antibody (goat anti-mouse) was labeled with FITC (Dianova, Hamburg, Germany) and used at a dilution of 1 : 50. Western blot analysis to determine cyclin D1 expression 56105 cells were plated per 60 mm2 culture dish and medium was changed the following day. After another 48 h cells were trypsinized, counted, washed twice in ice-cold PBS and frozen in liquid nitrogen. For protein preparation, aliquots of 26106 cells were lysed on ice in 200 ml lysis-buer (10 mM Tris-HCl pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate, 5 mM EDTA, 0.5 mM orthovanadate, 5 mM NaF, 50 mM pefa block, 10 mg/ml leupeptin and 2 mg/ ml pepstatin, antipain, aprotinine and chymostatine), centrifuged at 10 000 g for 15 min at 48C, and the supernatant was stored at 7708C. Protein concentrations were determined by the BioRad protein assay (BioRad, Munich, Germany). Samples (60 mg protein) were analysed under reducing conditions by SDS ± PAGE and blotted onto nitrocellulose membranes. Filters were blocked for 2 h in 10% low-fat milk in PBS/0.1% Tween-20 and incubated with anti-Cyclin D1 monoclonal antibody (1 mg/ml; NeoMarkers, Fremont, CA, USA) for 3 h at room temperature. After washing (56) with blocking buer, ®lters were incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (0.08 mg/ml; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) in blocking buer for 1 h at room temperature, washed again, and Cyclin D1 was detected by using the ECL reagent (Amersham, Braunschweig, Germany). Kinase assay Cells were lysed for 1 h on ice in lysis buer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20, 1 mM dithiothreitol, 10 mM b-

glycerolphosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 0.1 mM phenylmethylsulfonyl¯uoride, 2 mg/ml aprotinine, 10 mg/ml leupeptine, 50 mM pefa block), lysates cleared by centrifugation at 10 000 g for 20 min, and the protein concentration determined (Biorad). For immunoprecipitation, 200 mg protein of each sample was incubated (1 h) with protein G-Sepharose beads (equilibrated in lysis buer), incubated overnight at 48C with the cyclin D1 antibody DCS-11 (kindly provided by Dr Jiri Bartek, Copenhagen, Denmark), and washed with lysis buer and kinase buer (50 mM HEPES pH 7.5, 10 mM MgCl2, 2.5 mM EGTA, 10 mM b-glycerolphosphate, 1 mM NaF, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol). Thirty ml of kinase reaction mix (kinase buer with 20 mM ATP, 5 mCi [g-33P]ATP and 2 mg GST-pRB (amino acids 792 ± 928)) was added, samples were incubated at 328C for 30 min, boiled in SDS ± PAGE sample buer, and separated by electrophoresis in 10% polyacrylamide gels, followed by autoradiography. For preparation of the GST-pRB substrate, an E. coli culture transformed with pGEX-GST-pRB (amino acid 792 ± 928) was induced with IPTG and lysed as described by Matsushime et al. (1994). Fusion protein was bound to glutathione-Sepharose 4B and released by incubation with reduced glutathion. The concentration and purity of the soluble fusion protein was determined with the Biorad protein assay and by Coomassie blue staining of electrophoresed protein, respectively.

Acknowledgements We would like to thank H Steinbauer for excellent technical help in tumorigenicity studies, Dirk Breitkreutz for critical discussions, and Brigitte Plagens for stylistic improvements. We also wish to extend our thanks to Brigitte Engelhardt and coworkers for their excellent photo work. This work was in part supported by grants from EC (ENV4-CT96-01272 to P Boukamp) (Bio4 CT 96o464 to NE Fusenig), Sander Stiftung Nr. 98.013.1 (to P Boukamp), and the DFG (Bo 1246 3-1 to P Boukamp).

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