Overlapping loss of heterozygosity by mitotic ... - Europe PMC

Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7590-7594, September 1991

Medical Sciences

Overlapping loss of heterozygosity by mitotic recombination on mouse chromosome 7F1-ter in skin carcinogenesis (chemically induced tumors/suppressor genes/oncogenes)

ALBERT B. BIANCHI, NORA M. NAVONE, C. MARCELO ALDAZ, AND CLAUDIO J. CONTI* Department of Carcinogenesis, University of Texas, M. D. Anderson Cancer Center, P.O. Box 389, Smithville, TX 78957

Communicated by Michael H. Wigler, June 4, 1991 (received for review March 18, 1991)

chromosome 7 that includes, among others, the Harvey ras-) (Ha-ras-1) and the f3-globin (Hbb) genes (6). At least two putative suppressor loci have been assigned to the lip region: the Wilms tumor gene (WTI), mapped to iipi3 and assigned to mouse chromosome 2 (6), and the Beckwith-Wiedeman syndrome (BWS) locus on iip15.5, also associated with a second Wilms tumor locus (WT2) (7) and tightly linked to the Ha-ras-J and Hbb loci (6). Similarly, the region iipl5.5-pter has been identified (8) as the smallest segment of overlapping homozygosity attained by mitotic recombination in rhabdomyosarcoma, a pediatric tumor clinically associated with BWS. Recently, a third putative suppressor gene, the multiple endocrine neoplasia type 1 (MEN-i)-associated locus, has been mapped to the long arm of human chromosome 11 tightly linked to the int-2 gene (9, 10) on iiqi3. Embryonal rhabdomyosarcoma and MEN-1 have been proposed (8, 9) to conform to Knudson's two-hit model (11) of oncogenesis. Additional evidence supporting the presence on human chromosome 11 of one or more suppressor genes was provided by experiments showing complete suppression of tumorigenicity by microcell transfer of a single chromosome 11 in the human cervical carcinoma cell lines HeLa (12) and SiHa (13) as well as in a Wilms tumor-derived cell line (14). In experimental animal models, a putative suppressor locus was assigned (15) to the short arm of Chinese hamster chromosome 3, which shares extensive homology with human chromosome Up and mouse chromosome 7. Similarly, the involvement of a putative suppressor gene linked to the albino and Hbb loci on mouse chromosome 7 has recently been postulated (16) based on the shorter latency and increased incidence of AKR/J mice to chemically induced

A significant role for mouse chromosome 7 ABSTRACT abnormalities during chemically induced skin carcinogenesis has been advanced based on previous cytogenetic and molecular studies. To determine the frequency of allelic losses at different loci of chromosome 7 in skin tumors induced in the outbred SENCAR mouse stock by a two-stage initiationpromotion protocol, we compared the constitutional and tumor genotypes of premalignant papillomas and squamous cell carcinomas for loss of heterozygosity at different informative loci. In a previous study, these tumors had been analyzed for their allelic composition at the Harvey ras-) (Ha-ras-1) locus and it was found that 39% of squamous cell carcinomas had lost the normal Ha-ras-1 allele exhibiting 3 or 2 copies of the mutated counterpart or gene amplification. In the present study, by combining Southern blot and polymerase chain reaction fragment length polymorphism analyses, we detected complete loss of heterozygosity at the f-globin (Hbb) locus, distal to Ha-ras-i, in 15 of 20 (75%) skin carcinomas. In addition, 5 of 5 informative cases attained homozygosity at the int-2 locus, 27 centimorgans distal to Hb. Polymerase chain reaction analysis of DNA extracted from papillomas devoid of stromal contamination by fluorescence-activated sorting of single cell dispersions immunolabeled with anti-keratin 13 antibody revealed loss of heterozygosity at the Hbb locus, demonstrating that this event occurs during premalignant stages of tumor development. Interestingly, loss of heterozygosity was only detected in late-stage lesions exhibiting a high degree of dysplasia and areas of microinvasion. Analysis of allelic ratios by densitoof tumors that had become homozygous at Hbb metric s but retained heterozygosis at Ha-ras-I indicated mitotic recombination as the mechanism underlying loss of heterozygosity on mouse chromosome 7 during chemically induced skin carcinogenesis. These findings are consistent with the presence of a putative tumor suppressor gene linked to the Hbb locus in the 7FM-ter region of mouse chromosome 7, the functional inactivation of which may constitute a critical event in skin tumor progression, possibly during the malignant conversion stage.

thymic lymphomas. In the mouse skin carcinogenesis model, our laboratory previously identified (17) trisomy of chromosome 7 (Ts7) as one of the primary numerical chromosomal abnormalities found in the majority of severely dysplastic premalignant papillomas and squamous cell carcinomas (SCCs) induced by initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion with phorbol 12-myristate 13-acetate (PMA). Recently, we showed (18) that Ts7 occurs by nonrandom duplication of the chromosome 7 bearing a mutated Ha-ras-J allele and that -40% of SCCs presented loss of the normal Ha-ras-i allele exhibiting 3 or 2 copies of the mutated counterpart or gene amplification. In this regard, nondisjunction and/or mitotic recombination were among the mechanisms suggested by our (18) and other laboratories (19) to account for LOH on mouse chromosome 7 during skin tumor progression. The results reported here show that mitotic recombination is the major mechanism for reduction to homozygosity on this

Chromosomal deletions, nondisjunction, and mitotic recombination are among the mechanisms that may account for loss of heterozygosity (LOH), resulting in the unmasking of recessive mutations (1, 2). In this regard, the tumor-specific LOH at particular chromosomal loci has signaled the presence of putative suppressor genes in various human neoplasias (3, 4) and, in some instances, led to the cloning and identification of genes that were shown to be involved at defined stages of the tumorigenesis process (5). One region that has been shown to be affected by allelic losses in several human malignancies (3) is the short arm of chromosome 11 (lip), which shares a large syntenic group with mouse

Abbreviations: LOH, loss of heterozygosity; SCC, squamous cell carcinoma; DMBA, 7,12-dimethylbenz[a]anthracene; PMA, phorbol 12-myristate 13-acetate; RFLP, restriction fragment length polymorphism; cM, centimorgans. *To whom reprint requests should be addressed.

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chromosome and that SCCs exhibit a high frequency of LOH at loci distal to Ha-ras-J, with the smallest segment of overlapping reduction to homozygosity on the 7F1-ter region, distal to the Hbb locus. In addition, LOH at Hbb was detected in severely dysplastic papillomas, indicating that this event occurs in premalignant stages of tumor development. These findings strongly suggest that inactivation of a putative suppressor gene on chromosome 7F1-ter may represent a crucial event during chemically induced mouse skin tumor progression and might possibly be implicated in the acquisition of the malignant phenotype.

MATERIALS AND METHODS Animal Treatment and Tumor Short-Term Cultures. Tumors were induced on the dorsal skin of SENCAR mice (National Cancer Institute, Frederick, MD) by initiation with DMBA (10 nmol) and promotion with 2 Ag of PMA twice weekly, as described (20). Sections of the tumors were characterized histologically and the remainder was enzymatically dispersed at 370C during 40-50 min, as described elsewhere (18). The cell dispersion was centrifuged, resuspended in low calcium (50 AM calcium chloride) Eagle's modified essential medium (MEM) (21) supplemented with 1% fetal bovine serum, and plated at a density of 5 1x05-x x 106 viable cells per 25 cm2. After 24 hr, cultures were washed to eliminate unattached cells and harvested at 48-72 hr for DNA extraction. Cell Sorting of Papifloma Dispersions. Tumor tissue was enzymatically dispersed (18), filtered through a 50-pum nylon mesh (Bellco Glass), fixed through a series of graded ethanol concentrations (30%o, 50%, and 75%), and filtered once more through the 50-gm nylon membrane. The cell pellet was rinsed twice with phosphate-buffered saline solution (PBS)/ 0.1% bovine serum albumin and immunolabeled with monospecific anti-keratin 13 antibody (gift of D. Roop, Baylor College of Medicine, Houston, TX) as primary antibody and fluorescein isothiocyanate-conjugated anti-rabbit antiserum as secondary antibody (Vector Laboratories). Immunolabeled cells were then sorted out and quantitated in a fluorescence-activated cell sorting machine (Becton Dickinson, model 440) using standard operation procedures. Processing of Paraffm Sections, DNA Extraction, and Hybridization. These procedures were performed as described (18, 22). Recombinant DNA Probes. Probes used in this study were a 1600-base-pair (bp) Hha I fragment from plasmid pCR1:JBM9 (gift of B. A. Taylor, The Jackson Laboratory) for the mouse Hbb ,-globin gene cluster and a 3200-bp Sal I fragment from plasmid pSAM3.1 (gift of P. Stambrook, Univ. Cincinnati Medical Center, Cincinnati, OH) for the mouse adenine phosphorybosyltransferase (Aprt) gene. A segment of -250 bp was sequenced from probe int-2 i (gift of G. Peters, Imperial Cancer Research Fund Laboratories, St. Bartholomew's Hospital, London) to design the 3' amplimer used for amplification by polymerase chain reaction (PCR) of a 1600-bp fragment of the int-2 mouse gene. Densitometric Analysis. Grain density values (integrated optical density with background subtracted) of nonsaturated autoradiographic exposures were measured using a Biolmage Visage 60 densitometer (MilliGen/Biosearch, Novato, CA). Scans were normalized for DNA loading by rehybridization with a probe for the mouse Aprt gene located on mouse chromosome 8. A relative value of 1.0 was assigned to the densitometric ratio Hbb/Aprt as measured in control tissue DNA (spleen). PCR. For the Hbb locus, a two-allele polymorphism was detected by PCR using a three-primer strategy described previously (22). Briefly, one shared 5' amplimer (5'TAGGTGTGTAGATGTATCCA-3') and two 3' amplimers

Proc. NatL. Acad. Sci. USA 88 (1991)

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(5'-CAGCTGGGACAGAATTATAC-3' and 5'-ATGTATCTGCTACTTTCTAA-3') were used to generate amplification products of 500 bp and 600 bp corresponding to the HbbS and Hbbd alleles, respectively. After denaturation at 95°C for 1.5 min, each PCR cycle included 4 sec of primer annealing at 58°C, 60 sec of extension at 73°C, and 4 sec of denaturation at 94°C. A total of 28 cycles was performed in an Eppendorf thermocycler (model 6700-100; Eppendorf), followed by a final extension step at 73°C for 7 min. For the int-2 locus, a two-allele polymorphism (N.M.N., A.B.B., and C.J.C., unpublished data) was detected by amplification of a region located =600 bp downstream from exon 3 of the int-2 gene (23), which includes a 300-bp insertion. Thus, amplification by PCR generated products of =1900 bp and -1600 bp, named A1 and A2, respectively. The 5' amplimer (5'-CAGACCTTCCAGGGTTCTGT-3') was designed based on sequence data kindly provided by G. Peters (Imperial Cancer Research Fund Laboratories). The 3' amplimer (5'-ACAGCCACCACCTTGAGGTA-3') was designed based on data obtained by sequencing of the probe int-2 i. Primer annealing conditions were set at 60°C for 4 sec, followed by extension at 73°C for 72 sec and denaturation at 94°C for 4 sec, for a total of 28 cycles. PCR fragments were detected by electrophoresis on 1.2% agarose gels stained with ethidium bromide. Analysis of Ha-ras-1 Mutations by PCR. A 190-bp fragment flanking codon 61 of the Ha-ras-1 gene was amplified using oligonucleotides 5'-GACTCCTACCGGAAACAGGT-3' and 5'-AGGTGGCTCACCTGTACTGA-3' as 5' and 3' amplimers, respectively. After denaturation at 95°C for 1.5 min, each amplification cycle included 10 sec of primer annealing at 57°C, 60 sec of primer extension at 73°C, and 6 sec of denaturation at 94°C. A total of 30 cycles was performed followed by a final extension step at 73°C for 7 min. A -- T transversions on base 182 of codon 61 were analyzed by restriction of the PCR product with the enzyme Xba I and were detected as fragments of 69 and 121 bp by electrophoresis on 4% agarose gels.

RESULTS AND DISCUSSION Constitutional and tumor genotypes at loci on chromosome 7 were analyzed for LOH in skin tumors induced in SENCAR mice by initiation with DMBA and promotion with PMA. The outbred nature of SENCAR mice, widely used in two-stage skin carcinogenesis experiments, makes this stock suitable for analyses requiring constitutional polymorphisms. As shown in Table 1, tumors had previously been characterized (18) in terms of their normal/mutated Ha-ras-1 allelic composition based on the Xba I restriction fragment length polymorphism (RFLP) generated by a heterozygous A -- T transversion at codon 61 of the Ha-ras-1 gene (24, 25). Briefly, a Southern hybridization analysis of Xba I-restricted DNAs had revealed that -39%o of SCCs showed loss of the normal Ha-ras-1 allele with 2 or 3 copies of the mutated counterpart or gene amplification (18). DNAs had been extracted from short-term tumor cultures (24-72 hr) to eliminate contamination by stromal and inflammatory cells in the sample. To determine whether this reduction to homozygosity at the Ha-ras-1 locus, 32 + 2.2 centimorgans (cM) from the centromere (6, 26), resulted from either chromosomal nondisjunction or mitotic recombination, we sought to analyze tumor genotypes at other informative (i.e., heterozygous) loci on chromosome 7. The Hbb locus, 50 + 1.0 cM from the centromere (6, 27) and characterized (28) in the BALB/c and the C57BL/10 mouse strains by the Hbbd and HbbS alleles, respectively, was found to be polymorphic in the SENCAR mouse stock exhibiting an approximate allelic frequency of 0.54 for Hbbd and 0.46 for HbbS (n = 48) (A.B.B., N.M.N.,

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Table 1. LOH at the Hbb locus in chemically induced mouse skin carcinomas

Hbb Ha-ras-J Hbb/Aprt Pathology (normal/mutated) N T ratio 0 Spleen 2/0 1.0 1 SCC III 0/3 d/s d/-* ND 2 7967 SCC I 1/2 d/s -/s* 1.42 3 8041 SCC II 0/3 d/s d/1.34 4 7921 SCC IV 0/g.a. d/s d/1.43 5 7948 SCC IV 0/g.a. d/s 1.71 -/s 6 7977 SCC I 1/2 d/s d/1.39 7 7184 SCC II 0/3 d/st d/1.41 8 7231 SCC III 0/g.a. d/st d/0.83 9 7300 SCC I 1/2 d/st d/1.47 10 7302 SCC I 1/2 d/st d/1.50 11 7396 SCC I 1/2 d/s (d)/st 1.38 12 7525 SCC I/PAP 1/1 d/s (d)/st 0.93 13 7539 SCC II 1/2 d/s d/(s)t 1.33 14 7581 SCC II 2/0 d/s d/s ND 15 7431 SCC II 0/3 d/s d/1.61 16 7439 SCC I/PAP 1/1 d/st d/0.90 17 7798 SCC II n/m d/s (d)/st 0.90 18 7994 SCC II 0/2-0/3 d/s d/0.70 19 7232 SCC I 1/1 d/st d/ND 20 7765 SCC I n/m d/s ND d/Analysis of constitutional (N) and tumor (T) genotypes at the Hbb locus on mouse chromosome 7, as determined by Southern blot hybridization. Histopathology gradings and Ha-ras-J allelic compositions were obtained previously (18), with the exception of tumors 7798, 7994, 7232, and 7765. Hbb allelic dosages were calculated by densitometric comparison of the Hbb and Aprt (chromosome 8) autoradiographic signals, normalized with a normal spleen control Hbb/Aprt ratio of 1.0. PAP, papilloma; ND, not determined; g.a., gene amplification; n/m, normal and mutated Ha-ras-J alleles detected by PCR analysis without quantification (tumors 7798 and 7765). Samples 1-16 correspond to the following sample numbers in table 2 of ref. 18: 1-4, 9, 11, 15-17, 19-23, 25, and 26. Samples 17-20 were not included in ref. 18. *Based on relative autoradiographic intensity values, the faint signal corresponding to the lost allele may be attributed to the presence of minor stromal contamination. tConstitutional DNA not available for Southern blot analysis. Genotype determined by PCR analysis of DNA extracted from archival formalin-fixed paraffin-embedded tissue, as described (22). tParentheses denote reduced autoradiographic signal suggesting partial loss of the allele indicated.

Sample

Tumor Control 8066

and C.J.C., unpublished results). The Southern blots in Fig. 1 show the Xba I RFLP observed in constitutional (Fig. la) and tumor (Fig. lb) DNAs, after hybridization with the mouse Hbb probe pCR1/pM9. Tumor samples from informative cases show significant or complete loss of autoradiographic signal for the Hbbd or HbbS allele-derived restriction fragment pairs, indicating loss of heterozygosity. A summary of the Xba I RFLP analysis of DNA extracted from 20 SCCs, whose constitutional genotype was found to be heterozygous at the Hbb locus, is given in Table 1. The data show that LOH was seen at Hbb in 15 of 20 (75%) SCCs, as compared to 11 of 29 (39o) cases informative for Ha-ras-J. Interestingly, no tumors that retained heterozygosity at the Hbb locus were found to exhibit LOH at Ha-ras-J (Table 1). Conversely, 7 of 11 tumors heterozygous at Ha-ras-J exhibited complete reduction to homozygosity at Hbb, suggesting that the latter event is not necessarily related to loss of the normal Ha-ras-J allele but appears to be linked to a putative suppressor gene distal to Hbb. In addition, densitometric comparison (Table 1) between the Hbb (chromosome 7) and the Aprt (chromosome 8) (Fig. lc) hybridization signals indicated approximately the same allele copy number previously determined (18) at the D7Rp2 (13 cM from the centromere) (6) and the Ha-ras-1 loci, ruling out that homozygosity had developed as a result of a hemizygous deletion. One possible exception was found in tumor 7994, where the densitometric ratio calculated might be indicative of a deletion at Hbb. These combined results showing tumor-specific reduction to homozygosity on one marker (Hbb), retention of heterozygosity at a proximal locus (Ha-ras-J), and maintenance of allelic dosages provide strong evidence for mitotic recombi-

nation as the major chromosomal mechanism underlying LOH on mouse chromosome 7 during skin tumor progression and further support previous speculations (19) that the distal region of this chromosome may harbor a suppressor gene. To assess an apparent trend of increasing frequencies of LOH toward distal markers, we analyzed allelic losses at the int-2 locus, 74 ± 1.6 cM distal from the centromere (6, 29). A PCR strategy, similar to the approach previously described (22) to analyze LOH in small lesions and paraffin block sections, was used to detect a two-allele polymorphism generated by a 300-bp insertion in the 3' flanking region of the int-2 gene (N.M.N., A.B.B., and C.J.C., unpublished data). As shown in Fig. 2, five of five informative SCCs exhibited complete loss of constitutional heterozygosity, with three of five tumors remaining heterozygous at Ha-ras-1 (Table 2), providing additional evidence of mitotic recombination as a mechanism accounting for reduction to homozygosity on mouse chromosome 7. Although the inadequate number of markers available distal to Hbb prevented a precise assignment, the results reported here suggest that the putative suppressor locus under study is localized in the 7F1-ter region of mouse chromosome 7, to which int-2 and Hbb have been assigned (6). In complementary studies aimed to establish how allelic losses on chromosome 7 were associated with the premalignant evolution of mouse skin tumors, we analyzed 10 papillomas at different stages of development. As described previously (22), epithelial cell populations were sorted out from contaminating stromal and inflammatory cells by fluorescence-activated cell sorting of tumor single-cell suspensions labeled with an antibody against keratin 13, a type I

Up*

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Proc. Natl. Acad. Sci. USA 88 (1991) Table 2. LOH at the int-2 locus on mouse chromosome 7

a Constitutional genotype Lo

)D o

co 0 0)

r-

Hb b d -

CO

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Hbbs_

a))

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1

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Hbb int-2 Ha-ras-J Sample Tumor Pathology (normal/mutated) N T N T 1 8041 SCC II 0/3 d/s d/- A1/A2 Al 2 7948 SCC IV 0/g.a. d/s -/s A1/A2 A2 3 8045 SCC II 1/2 d/d d/d A1/A2 Al 4 7765 SCC I n/m d/s d/- A1/A2 A2 5 8962 SCC I n/m d/d d/d A1/A2 A2 PCR analysis of constitutional (N) and tumor (T) genotypes at the int-2 locus. Histopathology gradings and Ha-ras-I allelotypes were obtained previously (18), with the exception oftumors 7765 and 8962. Hbb genotypes were detected by Southern blot hybridization. g.a., Gene amplification; n/m, normal and mutated Ha-ras-1 alleles detected by PCR analysis without quantification (tumors 7765 and 8962).

r-

-

i

W .NW

i kb 4_10.6 --9.3 -8.6 -7. 1

b Tumor genotype

Hbb dHbb SI

-

a

"

w

stages. These observations strongly suggest that LOH on the distal part of chromosome 7 may be closely associated with malignant conversion in the transition between the papilloma and carcinoma phenotypes. Furthermore, the almost specific loss of the HbbS allele (13 of 15 SCCs and 3 of 3 papillomas) over the Hbbd counterpart suggests a possible linkage between the latter allele and the presumably inactivated copy of a suppressor locus. In this regard, it may be speculated that the affected allelic form either carried a recessive germ-line mutation or sustained a somatic alteration selectively due to differences in allelic mutability (32). Consistent with our findings is the fact that SSIN, an inbred mouse strain derived from SENCAR and characterized for its high incidence of papilloma formation but very low frequency of malignant conversion to carcinoma (33), exhibits the HbbS genotype, suggesting again a possible linkage between the latter allelic form and the wild-type allele of a putative suppressor gene hypothetically involved in the acquisition of the malignant phenotype. Although the small number of informative cases analyzed for the int-2 locus prevents us from assessing a similar association, tumorbearing SENCAR mice were found to be predominantly homozygous for the int-2 A2 allele as opposed to the int-2 Al genotype characteristic of SS1N, providing circumstantial evidence for a possible linkage between int-2 A2 and the presumably inactive allele of a suppressor locus. In previous studies, we determined that Ts7, found in the majority of severely dysplastic papillomas and SCCs (17, 34), resulted in an increase in the copy number of the mutated Ha-ras-J gene, mainly in the form of 1/2 and 0/3 (normal/ mutated) allelic compositions (18). In view of the results reported here, it may be speculated that inactivation of a putative suppressor locus on 7F1-ter complements Ha-ras-J activation during mouse skin carcinogenesis. In tumors exhibiting one normal and two mutated copies of the Ha-ras-J gene, a single mitotic recombination exchange at a crossover point proximal to Ha-ras-J may result in the simultaneous

C Aprt ---wmao-11

FIG. 1. LOH at the 3-globin Hbb locus on mouse chromosome 7. Matched sets of constitutional and tumor DNAs were analyzed by Southern blot hybridization. kb, Kilobases. (a) DNAs from normal tissue (spleen) digested with Xba I and hybridized to the Hbb DNA probe, pCR1:4BM9. DNAs 7812 and 7742 (underloaded) are constitutionally homozygous for the Hbb3 allele. (b) DNAs extracted from short-term (24-72 hr) cultures of skin carcinomas. Partial or complete LOH is revealed by the absence of the Hbbd (SCCs 7798, 7967, 7948) or HbbS (SCCs 8066, 8041-7921) allele-derived bands. (c) Southern blot in b rehybridized to a probe for the mouse Aprt gene (pSAM3.1) mapped to chromosome 8 for normalization of DNA loading.

keratin aberrantly expressed at early stages of papilloma progression (30, 31). In this regard, we previously determined (22) that detection of LOH events by PCR can be performed unequivocally in the presence of stromal DNA (i.e., genotypically constitutional) levels not exceeding 20% of the total tumor DNA, provided the amplification reaction is completed within the exponential range before reaching saturation at the plateau. As shown in Fig. 3, none of the four papillomas at 15 weeks of promotion (lanes 1-4) exhibited LOH at the Hbb locus. Nevertheless, loss of the HbbS allele was observed in one of four papillomas at 27-30 weeks (lanes 5-8) and in two of two tumors at 39 weeks (lanes 9-10) of promotion with PMA. Interestingly, each of the three latestage papillomas that exhibited allelic losses also presented a high degree of dysplasia with areas of microinvasion. Similarly, histopathological evaluation of SCC 7439 (Table 1), which exhibited complete loss of the HbbS allele, revealed large dysplastic areas graded as papilloma, suggesting that this event had occurred in the premalignant and malignant 7948 TN T

8041 N

int-2 A

int-2 A2

1-

7593

8045 7765 N T NT

8962 N T

up L

kb 2.0

Hbbd Hbbs

-1,0 1 8 - 506

-1 ~~~~~~~~~~1.6

FIG. 2. Constitutional (N) and tumor (T) genotypes of int-2 as determined by PCR analysis of DNAs extracted from short-term (24-72 hr) cultures of skin carcinomas. A two-allele polymorphism on int-2 was revealed by bands designated Al (-1900 bp) and A2 (-1600 bp). Loss of constitutional heterozygosity was demonstrated by the absence of the Al (7948, 7765, 8962) and A2 (8041, 8045) bands on tumor DNAs. L, 1-kb DNA ladder (BRL). The 1.2% agarose gel was stained with ethidium bromide.

FIG. 3. PCR analysis demonstrating LOH at the Hbb locus on mouse skin papillomas. Single cell dispersions from papillomas at 15 weeks (lanes 1-4), 27-30 weeks (lanes 5-8), and 39 weeks (lanes 9 and 10) of promotion with PMA were immunolabeled with antikeratin 13 antibody and sorted by fluorescence-activated cell sorting. Lanes 6, 9, and 10, papillomas showing loss of the Hbbs-derived band. Lanes 11 and 12, DNAs extracted from spleens homozygous for the HbbS (-500 bp) and the Hbbd (-600 bp) alleles, respectively. L, 1-kb DNA ladder (BRL). Fragments amplified by PCR were fractioned by agarose gel (2%) electrophoresis and detected by ethidium bromide staining.

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occurrence of two, and possibly three, events with strong selective advantages-namely, increased copy number of an activated oncogene, homozygosity for a presumably inactivated suppressor gene, and loss of the wild-type Ha-ras-) allele. Interestingly, the fact that most (8 of 11) advanced carcinomas (SCC II, SCC III, and SCC IV) (Table 1) exhibited complete LOH at Ha-ras-1 and Hbb provides evidence in favor of the strong progressional selective advantage presented by a recombination event encompassing both loci. Likewise, the observation that 7 of 9 well-differentiated carcinomas (SCC I) (Table 1) showed complete LOH at Hbb while still retaining the normal copy of the Ha-ras-) gene suggests that homologous recombination at a locus distal to but not including Ha-ras-) appears to be associated to a more differentiated phenotype, as opposed to moderately differentiated and anaplastic tumors, in which homozygosis was observed at all loci distal to and encompassing the mutated Ha-ras-) allele. Complementation between oncogene activation and loss of suppressor gene function has been advanced in various systems (4, 35). Among these, Syrian hamster tumors induced by activated v-Ha-ras and v-myc oncogenes were found to exhibit a nonrandom chromosomal loss (36), and tumor suppression by microcell transfer of a single chromosome was observed in transformed epithelial cells carrying activated ras genes (12, 13). Similarly, studies with Chinese hamster embryo fibroblasts (15) transfected with a mutant Ha-ras gene have shown a strong correlation between tumorigenicity, trisomy for chromosome 3q (harboring the Ha-ras gene), and loss of one and sometimes two copies of the corresponding short arm (3p), which shares extensive homology with human chromosome lip and mouse 7 and may harbor a suppressor gene. A similar interaction has also been found in human colorectal tumorigenesis, where the mutational activation of a ras gene [Kirsten ras (Ki-ras)] in early adenomas is complemented by multiple allelic losses

implicating known and putative suppressor genes (5). Among the molecular events postulated to mediate the conversion of ppiloma to carcinoma, previous studies (37, 38) have suggested a cooperation between the Ha-ras-1 and fos oncogenes in the acquisition of the malignant phenotype. In view of the role advanced for c-fos in malignant conversion and the evidence implicating the c-fos promoter region as a likely target for regulation by suppressor genes (39), it may be speculated that inactivation of a critical gene(s) may be involved in c-fos deregulation during this stage. Although the molecular events directly involved in the conversion stage of mouse skin tumors remain obscure, the findings reported here clearly support the notion that unmasking of putative recessive mutations through chromosomal mechanisms that confer homozygosity may be critically involved in this process. Additional studies may provide the basis to identify recessive genetic alterations directly involved in the multistage mouse skin carcinogenesis model. We thank G. Peters, P. Stambrook, and B. A. Taylor for providing the probes int-2 i, pSAM3.1, and pCR1:.BM9, respectively; D. Roop for the anti-keratin 13 antibody; D. Trono and A. Chen for excellent technical assistance; J. Chesnerfor assistance in histology; J. Ing and J. Riley for art work; and J. Mayhugh for typing this manuscript. This work was supported by the National Institutes of Health (Grants CA 42157 and CA 53123). 1. Knudson, A. G. (1986) Annu. Rev. Genet. 20, 231-251. 2. Hansen, M. F. & Cavenee, W. K. (1987) Cancer Res. 47, 4418-5527.

3. 4. 5. 6.

7. 8.

9. 10.

11. 12. 13. 14. 15.

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19. 20. 21.

22. 23. 24.

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