* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download A gene for the suppression of anchorage independence is located in
History of genetic engineering wikipedia , lookup
Genomic imprinting wikipedia , lookup
Gene expression programming wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Microevolution wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Designer baby wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Mir-92 microRNA precursor family wikipedia , lookup
Human–animal hybrid wikipedia , lookup
Hybrid (biology) wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Skewed X-inactivation wikipedia , lookup
Genome (book) wikipedia , lookup
Y chromosome wikipedia , lookup
Neocentromere wikipedia , lookup
A gene for the suppression of anchorage independence is located in rat chromosome 5 bands q22-23, and the rat alpha-interferon locus maps at the same region M. QUAMRUL ISLAM 1 , JOSIANE SZPIRER 2 , CLAUDE SZPIRER 2 , KHALEDA ISLAM 1 , JEAN-FRANCOIS DASNOY 2 and GORAN LEVAN 1 '* ' Department of Genetics, University of Gothenburg, Box 33031, S-400 33 Gothenburg, Sweden Department of Molecular Biology, Universite Libre de Bmxelles, Belgium 2 •Author for correspondence Summary Cell hybrids between malignant mouse hepatoma cells and normal rat fibroblasts -with approximately one set of chromosomes from each parent exhibited remarkable karyotypic stability. Most chromosomes of both parents were retained even after prolonged culture in vitro. Normally, such hybrids showed suppression of the transformed phenotype and formed no colonies in soft agar. However, two hybrids, BS140 and BS181, formed a few colonies in soft agar when many cells were seeded, and also occasional foci of cells were detected piling up in monolayer cell cultures. We isolated soft agar colonies (a-subclones) and subclones from foci (h-subclones) of both hybrids, and, as a control, subclones of cells from random areas without foci of one hybrid (BS181 p-subclones). When tested for soft agar growth, cells from the aand h-subclones of both BS140 and BS181 formed colonies at frequencies comparable to the malignant mouse hepatoma parent, whereas the control cells of the BS181 p-subclones (like the normal rat parental cells) yielded no soft agar colonies. All the cell lines -were subjected to detailed karyotype analysis in G-banding, which resulted in the finding that cells from the original BS140 hybrid contained at least one copy of each rat chromosome, whereas BS140 a- and h-subclones had lost both copies of rat chromosome 5. Similarly, the original BS181 hybrid contained at least one copy of each rat chromosome, whereas BS181 a- and h-subclones displayed a deletion of the segment q22-23 of rat chromosome 5. In contrast, the control BS181 p-subclones contained one or two copies of non-deleted rat chromosome 5. The conclusion is that a gene for the suppression of anchorage independence is located in the segment 5q22-23. We propose to call this gene SAI1 (for suppression of anchorage independence). Using Southern blotting, we tested whether any of several gene probes, known to correspond to DNA sequences in rat chromosome 5, were homologous to sequences in the deletion. Only one probe, corresponding to the active alphai-interferon gene, was shown to be located within the deletion. Hence, the SAI1 gene is closely linked to the alphai-interferon gene, and might be identical to this locus. Introduction hereditary cancers such as retinoblastoma (Knudson, 1985), where both normal alleles have to be inactivated for the malignant phenotype to be expressed (Cavenee et al. 1983). The occurrence of emerogenes has also been inferred from another line of research, i.e. the study of hybrids between malignant and normal somatic cells. Many workers have shown that in such hybrids the tumorigenic phenotype is suppressed, as determined by lack of ability to form tumours in syngeneic animals or in nude mice (Harris et al. 1969; Wiener et al. 1971, 1974; Stanbridge, 1976; Marshall & Dave, 1978; Sager & A group of genes with the ability to suppress the malignant phenotype in mammalian cells has been identified. These genes have been called anti-oncogenes or tumour-suppressor genes, and the inactivation of both alleles of such genes seems to be a requirement for malignant transformation in many instances. Following the suggestion of Todaro (1988), we will call these genes emerogenes. The dominant action of normal alleles of emerogenes is convincingly illustrated by the study of Journal of Cell Science 92, 147-162 (1989) Printed in Great Britain © The Company of Biologists Limited 1989 Key words: tumour suppression, emerogene, anchorage independence, alpha-interferon. 147 Kovac, 1978; Stanbridge et al. 1981, 1982; Benedict et al. 1984). It has usually been possible, however, to isolate, from the suppressed hybrid population, single cells that have regained the tumorigenic phenotype. Such revertants display lower chromosome counts than the original hybrid and it has been concluded that the reversion is due to loss of chromosomes that carry tumour suppressor genes (emerogenes); these studies led to the conclusion that such genes are located on mouse chromosome 4 (Jonasson et al. 1977; Evans et al. 1982), on Chinese hamster chromosome 3 (Craig et al. 1988) and on human chromosomes 1 and 11 (Benedict et al. 1984; Kaelbling & Klinger, 1986; Srivatsan et al. 1986; Saxon et al. 1986). This field has recently been reviewed by several authors (Sager, 1985, 1986; Stanbridge, 1985; Harris, 1985, 1986; Klein, 1987). In vitro studies have shown that, at least in some hybrids, transformation-associated properties like anchorage independence are also suppressed in hybrids formed between transformed, malignant cells and normal cells (Marshall & Dave, 1978; Marshall, 1980; Szpirer & Szpirer, 1980; Marshall & Sager, 1981; Dyson et al. 1985; Stoler & Bouck, 1985). However, tumorigenicity and transformation can be dissociated, since some hybrids formed between tumour and normal cells remain transformed but are non-tumorigenic (see e.g. Straus et al. 1976; Stanbridge & Wilkinson, 1978). Obviously, it is of great interest to identify the chromosomes that carry tumour- or transformation-suppressor genes. Most investigations in this field have been carried out with intraspecific hybrids (Wiener et al. 1971; Sager & Kovac, 1978; Klinger, 1980; Stanbridge et al. 1981). In these studies a major problem has been to distinguish unequivocally between the chromosomes of the abnormal and of the normal parental cells. In order to circumvent this problem various approaches have been attempted, such as utilizing translocation chromosomes, variations of C-bands among strains, isozyme variations, or restriction-fragment length polymorphisms (RFLPs). Of necessity, these methods focus on but one or a few of the chromosomes. From the viewpoint of refined chromosome identification, interspecific hybrids should be a much more workable material, but in this case, there is often the disadvantage of karyotype instability in the hybrids and selective loss of chromosomes from one of the parental species. In the present investigation, we have studied interspecific hybrids between malignant, transformed mouse cells and normal embryonic rat skin fibroblasts. These hybrids proved to have remarkably stable chromosome constitutions, and therefore provided a means of determining the derivation of all the chromosomes whether from the malignant or the non-malignant parental cell. Hybrids containing approximately one set of chromosomes from each of the parental cells were shown to be suppressed for the transformed phenotype, i.e. to be anchorage-dependent. From the primary, non-transformed hybrids, anchorage-independent sublines with limited chromosome loss could be isolated. The chromosome analysis of these hybrids indicated clearly that a suppressor of anchorage independence was located in a 148 M. Q. Islam et al. specific region of rat chromosome 5. We also showed that the rat alpha-interferon locus was located in the same region. Materials and methods Parental and hybrid cells The BWTG3 cell line is a clonal derivative from a hepatoma, which arose spontaneously in a mouse of the C57BL/J strain. The BWTG3 clone was isolated after selection in 6-thioguanine and is deficient in the HGPRT enzyme. Rat skin fibroblasts (RSF) were obtained from skin of embryos of the SpragueDawley strain and were cultured for about 2 weeks before fusion. The derivation of the BS series of mouse-rat hybrids has been described (Szpirer & Szpirer, 1979). Briefly, mouse BWTG3 and rat skin fibroblasts were harvested by trypsinization, rinsed in culture medium without serum, and fused in suspension using u.v.-irradiated Sendai virus. Hybrids were selected in HAT-medium (Littlefield, 1964). In this medium BWTG3 cells are effectively killed. The normal rat fibroblasts do not form any distinct colonies and the HAT-resistant hybrid clones (one per dish) could be isolated by means of stainless steel cylinders about 2 weeks after fusion. Growth in soft agar Cells were tested for growth in medium containing 0 - 3 % agar (Noble agar, Difco) by the method of MacPherson & Montagnier (1964). Two or three replicate plates were inoculated with 102—106 cells. The dishes were fed every week with 1 ml of medium. Colonies larger than 0-1 mm across were counted after 4 weeks of incubation at 37°C in a humidified CO2 incubator. As a control, the plating efficiency of cells tested for growth in agar was determined by plating 10 —10 cells in 5 ml of medium in 60 mm dishes: 1 ml of medium was added to each dish after 7 days, and the number of colonies was counted after about 2 weeks. Chromosome analysis Chromosome preparations were made and G-banded according to the standard techniques of our laboratory (Martinsson et al. 1982; Islam & Levan, 1987). Complete karyotypes were arranged from cutout chromosomes of enlarged photographic prints. At least 10 cells were karyotyped from each line studied. The mouse chromosome nomenclature recommended by the Committee on Standardized Genetic Nomenclature for Mice (1972) and by Nesbitt & Francke (1973) was followed. For rat chromosomes, the chromosome nomenclature recommended by the Committee for a Standardized Karyotype of Rattus norvegicus (1973) and the G-banding nomenclature of Levan (1974) were applied. Southern blot analysis This was done as described by Southern (1975). The probes were labelled according to the method of Feinberg & Vogelstein (1984), using the multiprime DNA labelling system (Amersham). The alpha-interferon probe was the pPCl plasmid, containing a 2-Skb EcoRl fragment, which includes the rat alphapinterferon gene (Dijkema et al. 1984). Results General characteristics of BS cell hybrids Two types of hybrids emerged from the cell fusion experiments between BWTG3 mouse hepatoma cells and normal rat skin fibroblasts. Type I hybrids contained about 100 chromosomes representing approximately the sum of the chromosome numbers of the parental cells less 10% (Szpirer & Szpirer, 1979). Specifically, the average number of rat chromosomes in these hybrids was between 32 and 38. As has been described (Szpirer & Szpirer, 1980) the type I BS hybrids resembled the nontransformed rat fibroblast parents in that they grew to a relatively low saturation density and they did not form colonies in soft agar: they clearly exhibited suppression of the transformed phenotype. Type II displayed higher total chromosome numbers (averages between 122 and 193 in different type II hybrids) and also fewer rat chromosomes (averages between 12 and 27). The relative contribution of genetic material from the hepatoma mouse parent was thus considerably greater in the type II hybrids, and they proved to grow to higher saturation densities on plastic and to have cloning efficiencies in soft agar that resembled that of the BWTG3 cells; these hybrids were not suppressed for the transformed phenotype (Szpirer & Szpirer, 1980). Growth characteristics of two type I hybrids: BSJ40 and BS181 As stated above, most type I hybrids were unable to form colonies in soft agar. Two hybrids, BS140 and BS181, however, yielded a few soft agar colonies when many cells were plated. Such rare colonies were isolated and propagated separately (BS140 a-series and BS181 a-series). Furthermore, in these two hybrids foci of cells piling up could be distinguished in confluent culture bottles. Such foci of high cell density were isolated, subcloned, and propagated separately (BS140 h-series and BS181 h-series). In addition, as a control, BS181 cells were subcloned on plastic, and subclones picked at random (BS181 p-series). Each of the resulting lines was tested for growth in soft agar, and it was found that the a-series as well as the h-series of subclones would form colonies in soft agar at frequencies comparable to that of the malignant mouse parent, whereas cells from the p-series were completely unable to form such colonies. The results have been compiled in Table 1. Chromosome analysis General findings. In order to determine whether the transformed a- and h-subclones derived from BS140 and BS181 had lost specific chromosomes, the chromosome constitution of these cells was determined in detail and compared with that of the parental, non-transformed hybrids and with that of the control, non-transformed subclones of the BS181 p-series. At least 10 complete karyotypes were analysed from each cell line studied. Average total chromosome numbers as well as numbers of mouse- and rat-derived chromosomes are listed in Table 2. From this table it is evident that the loss of chromosomes was quite limited, and that chromosomes had been lost from both parental cell types to approximately the same degree. Tables 3 and 4 give data on the average number of each mouse chromosome per cell: normal mouse chromosomes in Table 3 and mouse- Table 1. Cloning efficiency of the mouse and rat parental cells and of the derived cell hybrids Cloning efficiency Cell line Parents BWTG3 RSF Hybrids BS140 On plastic (%) In soft agar (%) 30 2xlO~ 3 9 <10~ 5 19 <5xl0~5 0-05 1-5 0-15 1 5 2 BS140hl BS140h2 BS140h3 BSHOalO BS140a20 BS140a31 20 13 25 15 30 25 BS181 BS181H1 14 BS181a2 BS181a3 BS181a4 BS181a5 BS181plO BS181pll 35 30 25 10 22 40 10 2xlO~ 3 3 10 0-2 1 5 <2xlO-4 <2xlO-4 derived markers in Table 4. In Table 5 the average numbers of rat chromosomes per cell have been recorded. Since the occurrence of rat-derived marker chromosomes in these lines was quite limited, we have not made a separate table for them. In Table 5 these markers have been included among the normal chromosomes from which they were derived. The kinds of rat chromosomal markers found were translocations, deletions and duplications. Most of them were seen in single cells only. A detailed description of the rat markers is given in Table 5. Loss of rat chromosome 5 material in the anchorageindependent sublines. The cells of the BS140 hybrid clone contained at least one copy of each mouse and rat chromosome (Fig. 1). The anchorage-independent subclones of BS140 (the h- and a-series) had very similar karyotypes except for one significant deviation: they contained no copy of rat chromosome 5. Sample karyotypes from BS140h3 and BS140a20 are given in Figs 2 and 3. The cells of the BS181 hybrid clone also contained at least one copy of every mouse and rat chromosome (Fig. 4). All hybrids of the BS181 h-series, a-series and p-series of subclones had karyotypes similar to that of the original BS181 clone. In this case there was no systematic loss of any rat chromosome from the h- and a-series of subclones: instead, it was detected that each of these subclones had undergone a specific chromosomal change: a deletion of rat chromosome 5. Examples of complete karyotypes of BS181hl and BS181a3 are given in Figs 5 and 6. In contrast, cells of the non-transformed BS181 subclones of the p-series displayed one or two copies of rat chromosome 5 with no signs of deletion. The fact that the five h- and a-subclones isolated independently from BS181 were all characterized by a deleted rat chromosome 5 raises the question of whether the deletion was the same in the different subclones. In Suppressor gene in rat chromosome 5 149 Table 2. Average number of mouse- and rat-derived chromosomes in the parental cells, and in the mouse-rat cell hybrids BS140 and BS181 and derived lines Average number of chromosomes (range) Parents BWTG3 RSF Total Rat Mouse Cell line 42 66-7 (63-70) 42 66-7 (63-70) Hvbrids BS140 60-1 (53-67) 56-3 (52-62) 62-0 (59-66) 61-0(56-65) 35-7 (32-40) 95-8 (89-101) BS140hl BS140h2 BS140h3 31-7(29-34) 31-1 (29-34) 30-6 (28-35) 88-0 (83-96) 93-1 (90-96) 91-6(88-96) BS 140a 10 BS140a20 BS140a31 61-8 (60-64) 55-4 (51-62) 57-6 (54-60) 27-6 (26-30) 33-1 (31-35) 29-6 (27-31) 89-4 (87-93) 88-5 (83-94) 87-2 (83-91) BS181 BS181hl 61-9 (55-67) 36-6(31-41) 98-5 (86-103) 58-6 (53-62) 35-9 (33-39) BS181a2 BS181a3 BS181a4 BS181a5 56-1 55-5 54-4 52-2 57-9 59-9 320 39-4 36-1 36-6 40-0 38-4 94-5 88-1 94-9 90-5 88-8 97-9 98-3 BS181plO BS181pll (48-61) (53-59) (46-58) (44-58) (41-63) (51-71) (26-36) (36-43) (32-40) (35-39) (30-48) (34-40) (90-99) (74-97) (91-100) (84-94) (80-93) (71-111) (90-107) q22-23 of rat chromosome 5. We propose to call this gene SAI1 (for suppression of anchorage independence). It was observed that in the transformed BS140 h- and a-sublines, the loss of rat chromosome 5 was accompanied by an increase of mouse chromosome 4 (Tables 3 and 5). No corresponding increase was seen in the non-transformed BS181 p-sublines (which retained rat chromosome 5) or in the transformed BS181 a- and p-sublines (which retained a deleted rat chromosome 5). Obviously, this may be purely fortuitous, but since mouse chromosome 4 appears to be largely homologous to rat chromosome 5 on the gene level (see Discussion), Fig. 7, instances are presented of rat chromosome 5 from BS181 and the five BS181 h- and a-subclones together with a diagrammatic representation of a normal rat chromosome 5. It is evident without any doubt that the segment deleted in each of the five sublines was the same, namely band 5q22-23. The combined information in Tables 1 and 5 makes it obvious that recovery of anchorage independence in the hybrids is correlated with loss of chromosomal material from chromosome 5 of the normal parental rat cell (Table 6). The inevitable conclusion is that a suppressor of the transformed phenotype is located in the segment Table 3. Average number per cell of each individual mouse chromosome in parental BWTG3 cells and in the mouse-rat cell hvbrids BS140 and BS181 and derived sublines Mouse chromosome X 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Marker 1-0 1-9 3-8 2-5 2-0 2-0 4-0 3-1 20 2-9 3-0 4-6 2-7 2-9 2-1 4-7 3-0 3-0 4-7 2-5 8-2 0-9 2-7 2-5 2-8 2-7 3-1 1-8 1-9 4-3 3-6 4-3 3-4 2-7 0-5 0-7 0-8 1-8 1-8 0-9 1-6 1-6 2-4 2-7 4-8 4-0 4-4 2-8 3-1 3-0 30 2-8 2-8 1-0 1-1 1-1 1-0 10 0-9 3-9 5-0 4-8 4-8 3-8 3-8 1-8 0-9 0-7 0-9 0-9 0-8 2-1 3-4 2-0 2-6 21 2-9 1-9 2-2 1-9 1-2 1-2 1-8 1-5 3-2 2-4 2-5 2-5 2-9 1-9 3-0 3-9 4-4 1-6 1-1 1-3 1-1 1-5 1-1 11 0-9 1-2 3-6 3-0 2-4 3-3 3-1 1-0 4-4 4-0 2-2 3-4 3-2 0-6 2-6 BS181plO BS181pll 0-9 1-0 1-6 1-9 2-0 2-8 2-3 2-8 1-8 1-2 11 11 3-5 4-8 2-6 31 2-0 1-5 2-7 3-0 2-0 2-0 2-3 2-3 2-1 2-0 0-9 1-8 20 3-4 1-8 1-6 1-8 1-8 1-7 1-8 1-0 1-1 3-2 3-4 1-9 1-8 1-2 1-0 3-8 4-3 2-6 2-6 2-8 3-5 2-2 1-8 1-3 1-8 11 1-5 1-2 2-2 2-5 2-1 3-0 2-7 2-9 1-9 2-5 8-8 8-8 13-3 10-5 11-9 9-8 9-7 2-9 2-9 21 2-9 30 2-8 1-0 0-9 2-0 1-9 1-1 2-8 1-9 2-0 2-5 20 3-7 2-8 3-1 1-9 1-9 2-0 0-9 1-7 2-8 1-6 2-4 2-4 1-1 2-6 1-0 2-0 2-0 2-2 2-1 1-8 2-2 2-9 2-2 2-7 30 4-4 3-6 1-5 3-4 2-9 2-7 2-8 2-4 2-1 3-6 4-0 5-0 4-4 4-2 3-2 4-0 3-4 3-4 4-5 4-8 41 3-7 3-2 4-9 4-2 4-5 4-0 4-6 5-0 2-2 4-2 4-8 5-3 1-7 1-2 1-9 1-5 10 10 0-9 BS181 BS181hl BS181a2 BS181a3 BS181a4 BS181a5 2-6 2-2 2-4 2-3 2-8 1-9 30 3-4 4-2 3-7 3-7 2-9 3-5 2-6 4-5 5-1 4-7 1-7 10 10 1-0 1-1 0-9 1-0 1-0 0-9 1-8 1-0 0-7 0-8 0-7 0-9 0-9 0-4 1-1 0-7 1-1 11 0-9 Cell line Parent BWTG3 Hybrids BS140 BSHOhl BS140h2 BS140h3 BS140al0 BS140a20 BS140a31 150 1-0 10 11 M. Q. Islam et al. 2-6 11 1-7 2-2 0-7 1-8 2-8 2-7 2-7 2-8 2-6 2-8 3-3 3-0 3-6 3-5 3-6 3-4 12-4 10-6 10-0 7-5 14-3 8-8 12-7 9-2 Table 4. Average number per cell of recurrent mouse-derived marker chromosomes in the parental mouse BWTG3 line and in the hybrids BS140 and BS181 and derived cell lines Marker chromosome* Cell line Parent BVVTG3 Hybrids BS140 BSHOhl BS140h2 BS14Oh3 marl mar2 mar3 mar4 mar5 mar6 1-0 1-0 1-0 — — _ 1-0 0-9 0-7 0-7 0-6 3-0 1-6 0-8 1-8 1-8 2-0 0-9 0-8 0-9 10 1-1 1-4 0-8 1-0 1-8 0-1 0-9 0-6 0-9 0-4 1-7 1-2 2-1 1-1 1-5 1-2 0-9 1-0 11 1-0 10 1-0 1-3 0-9 1-0 0-9 1-0 0-6 0-7 BSHOalO BS140a20 BS140a31 0-9 1-0 0-9 0-8 BS181 BSlSlhl 1-1 - 0-8 0-7 BS181a2 BS181a3 BS181a4 BS181aS 0-7 0-8 0-3 0-1 0-1 BS181plO BSlSlpll 10 0-7 0-8 10 1-2 1-S 1-2 0-9 0-7 - mar7 mar9 marlO 01 0-4 0-9 0-7 0-6 1-6 - 0-1 0-2 - : Only the most common and clearly identifiable markers have been listed; a number of other markers were also seen sporadically. *The marker chromosomes listed mostly represent Robertsonian translocations and have the following compositions: marl, rob(l; 1); mar2, rob(l;8); mar3, 15p+; mar4, rob(10;10); mar5, rob(6;6); mar6, rob(17;17); mar7, rob(8;13); mar8, rob(8;17); mar9, rob(9;17); marlO, rob(12;12). Actual pictures of these markers can be seen in Figs 1-6. Table 5. Average number per cell of each individual rat chromosome in mouse-rat hybrids BS140 and BS1SJ and derived cell lines Rat chromosome Hybrid line X Y - 2-4 1- 2-1 1-8 1-3 1-9 1-9 1-S — - 1-0 •1 •6 ; •8 ; •0 : •8 2-3 2-1 1-4 1-9 10 1-5 2-0 20 1-5 1-6 1-8 1-6 1-1 1-8 >9 1-0 1-8 1-0 1-0 •0* 2-0 1-9 2-1 2-0 1-9 21 1-9 2-0 1-9 2-3 1-8 1-0 2-0 1-9 1-9 2-1 1-9 1-8 0-9 1-6 1-6 2-1 1-0 1-0 0-9 1-1 1-0 1-7 1-9 1-8 1-8 1-7 1-8 2-0 2-0 — — ()-9 1-9 1-9 1-8 1-0 2-0 10 1-2 0-9 11 1-9 1-8 1-1 1-1 1-8 1-7 \-2 1-7 M 1-9 1-9 3-0 2-0 2-2 2-0 •6 •6 2-6 1-0 1-9 1-9 BS140 2-0 BSHOhl BS140h2 BS140h3 1-9 2-0 1-9 BSHOalO BSH0a20 BS140a31 BS181 BS181a2 BS181a3 BS181a4 BS181aS 1-9 1-9 1-9 1-0 1-0 1-0 1-0 1-0 1-0 1-0 1-0 1-0 1-0 1-0 1-0 BS181plO BS181pll 1-0 1-1 0-7 10 BSlSlhl •8 1-1 •1 •0* •0* •0* •0* 10 11 12 13 14 15 1-7 1-6 1-2 1- 1-0 11 0-9 1-8 1-7 1-8 1-0 1-0 1-0 1-6 1-3 1-2 1-6 1-2 1-1 1-1 1-3 1-3 20 1-3 1-7 11 1-0 1-0 11 0-9 1-0 2-0 1-0 1-2 1-8 1-0 10 2-5 2-7 2-9 2-4 2-8 1-0 10 1-0 1-9 2-7 1-9 2-9 1-8 2-1 1-9 2-0 2-0 1-9 1-7 1-8 2-1 1-8 1-9 1-0 1-9 1-3 1-3 1-2 10 1-0 1-1 0-9 1-0 2-0 0-5 1-5 1-8 1-8 1-9 1-4 1-9 1-8 1-0 1-8 1-9 1-8 0-9 2-0 2-0 20 1-3 1-2 1-8 1-9 1-6 1-9 2-0 1-7 2-5 1-5 2-0 1-8 20 2-0 21 1-9 1-9 1-8 2-0 1-9 1-6 1-7 1-8 1-9 2-1 16 17 18 19 20 1-5 1- 1-5 2-1 1-9 2-9 2-1 2-2 1-9 1-6 1-3 11 1-3 1-3 1-0 1-8 1-9 1-5 1-8 1-9 1-3 3-5 2-1 1-8 2-4 1-6 1-9 3-2 2-8 1-0 1-8 10 1-6 1-8 1-5 1-8 2-0 2-0 0-8 1-0 11 1-7 0-5 1-4 2-1 2-1 2-1 1-7 11 1-6 1-9 2-0 \-2 1-7 1-7 1-9 10 1-9 1-0 1-7 2-7 1-2 2-6 1-1 1-8 1-9 2-4 1-5 1-9 1-8 2-1 1-9 1-9 1-5 1-6 Rat marker chromosomes have been scored among the normal chromosomes they were derived from. As mentioned in the text, rat-derived chromosome markers occurred very sparsely. Often they were seen in single cells only (6 instances). Deletion of the short arm of chromosome 3 was seen occasionally, and (possibly as a consequence of a 3p deletion) Robertsonian translocations involving both copies of chromosome 3 (5 instances) or chromosome material added at the centromere of chromosome 3(11 instances). None of the aberrations involving breakage at the centromere of chromosome 3 was seen consistently in any of the lines. Only two cases of rat-derived translocation chromosomes were seen regularly in the majority of the cells of specific lines: (1) a chromosome derived from a translocation event involving chromosomes 3 and 4, namely der(4) (4cen-4q22::3ql2-3qter), was present in 9 out of 12 cells analysed from BS181a2 (scored as chromosome 4 in the Table); (2) in all the subclones of the BS140 h- and a-series each copy of chromosome 11 displayed additional chromosome material located terminally in the short arm. This material could not be identified unequivocally, but the G-banding pattern (or rather lack of banding) was consistent with that of the terminal segment of rat chromosome 5 (bands 5q35-qter). *The number S chromosomes marked by an asterisk display a deletion of the region q22-23. Suppressor gene in rat chromosome 5 151 Mouse M t 21 i» s,< in isis a 6 7 8 9 W 11 12 13 14 15 W ** 4A/f A£A i I tkv 17 lit 18 19 i v 16 r r ^ # e B C 0 E F G H I J K Rat !!!! ft M • I 15 1« _ _ ft 11 12 •« Sf M 17 .__.«_ W_ 13 14 «« 20 XX Fig. 1. Karyotype of cell from the anchorage-dependent hybrid BS140. Mouse-derived marker chromosomes: A, rob(l;l); B, rob(l;8); C, rob(10;10); D, 15p+; E-K, unidentified. Note presence of two normal copies of rat chromosome 5. (The presence of 4 copies of rat chromosome 1 was only seen in this particular cell.) 152 M. 0. Islam et al. Mouse it a s iitft i% 62 Ii 11 12 13 14 16 17 18 19 E F G Ii 15 H I J K L M Rat ! )) n I • 9 10 11 11 13 I ii a ii if . $f U 16 ,17 . 1* If 20 XX . 14 _.M Fig. 2. Karyotype of cell from the anchorage-independent hybrid subline BS140h3, isolated from cell-dense focus in BS140. Mouse-derived marker chromosomes: A, rob(l;l); B, rob(l;8); C, rob(10;10) (2 copies); D,E, ISp-l- (2 copies); F, rob(6;6); G, rob(3;7); H, rob(17;17); I-M, unidentified. Note absence of rat chromosome 5. Suppressor gene in rat chromosome 5 153 Mouse It? 3 i ft* 7 4 13 • ftff III t 15 » « C fi 18 17 10 14 12 11 9 19 » . . . H Rat I I 1 I 8 I 15 n 4 f 10 i 3 Mr 11 18 f 12 14 20 ii Fig. 3. Karyotype of cell from the anchorage-independent hybrid subline BS140a20, isolated in soft agar from BS140. Mousederived marker chromosomes: A, rob(l;8); B, rob(10;10); C, 15p+; D, rob(6;6); E, rob(8;13); F - K , unidentified. Note absence of rat chromosome 5. (The translocation between rat chromosomes 1 and 13 was only seen in this particular cell.) 154 M. Q. Islam et al. Mouse im i •< i Hi I 10 it I; 13 14 il 15 UM t o 16 It ( It X mlr... A B C D E F G H Rat M II I )) it it a u 10 11 15 If n u 12 I! 17 II U 13 • 19 If f 20 U 14 U X Y Fig. 4. Karyotype of cell from the anchorage-dependent hybrid BS181. Mouse-derived marker chromosomes; A, rob(l;8); B, rob(10;10); C, 15p+; D, rob(6;6); E, rob(15;18); F - H , unidentified. Note the presence of a normal copy of rat chromosome 5. Suppressor gene in rat chromosome 5 155 Mouse { i*i llili *•«» i 4 if n ;; ? a 13 u IH ;W 16 17 1« H 11 :c 12 I n t X i I I H U I It* i» I ». :t •• • • 10 11 12 13 14 15 IS 17 It 20 X Y 19 Fig. 5. Karyotype of cell from the anchorage-independent hybrid subline BS181hl, isolated from cell-dense focus in BS181. Mouse-derived marker chromosomes: A, rob(l;8); B, rob(10;10); C, 15p+; D, rob(14;17); E, unidentified; F, rob(7;13); G-J, unidentified. Note presence of an interstitial deletion in rat chromosome 5. 156 M. 0. Islam et al. Mouse t iti IKS H» %a * » 5 12 13 14 15 l*» I 11 Mil 16 17 B C 18 D E 19 F JC 6 Rat J 1 1* ! 2 121 8 i» 15 3 fti It 1 1 fA 11 12 4 5 9 10 M ift 1- 11 17 18 It If 11 6 u 13 20 «% 14 X Y j Fig. 6. Karyotype of cell from the anchorage-independent hybrid subline BS181a3, isolated in soft agar from BS181. Mousederived marker chromosomes: A, rob(l;l); B, rob(10;10); C, 15p+; D - G , unidentified. Note presence of interstitial deletion in rat chromosome 5. Suppressor gene in rat chromosome 5 157 RN05 BS181 BS181 hi a2 a4 a3 a5 Fig. 7. Diagram of rat chromosome 5 and cutout instances of rat chromosome S from the anchorage-dependent hybrid BS181 and anchorage-independent sublines, the latter exhibiting the interstitial deletion of bands q22-23. Table 6. Correlation of loss of chromosome 5 material from the normal rat parent and anchorage-independent groivth in the mouse-rat hybrids Hybrid line Rat chromosome 5 Anchorage dependence BS140 Present Dependent BSHOhl BS140h2 BS140h3 BSHOalO BS140a20 BS140a31 Absent Absent Absent Independent Independent Independent Absent Absent Absent Independent Independent Independent BS181 Present Dependent BSlSlhl BSl8la2 Deleted Deleted Deleted Deleted Deleted Present Present Independent Independent Independent Independent Independent Dependent Dependent BS181a3 BS181a4 BS18la5 BS181plO BS18lpll the observed increase in mouse chromosome 4 may be required to compensate for the loss of some housekeeping gene functions on rat chromosome 5. The rat Sc/22-23 region contains the alpha-interferon locus The assignment of known genes to the rat 5q22-23 region would provide 'landmarks' useful, for instance, for chromosome walking experiments aimed at identifying the SAI1 gene, or for comparative mapping with the human genome. Several genes have been assigned by us (unpublished data) and by others to rat chromosome 5. Using gene-specific probes in Southern blot analyses, we have determined whether any of them was included in the deletion present in the BS181 a- and h-hybrids. Among the loci tested, a single one, namely the alpha-interferon locus, was found to map in the 5q22-23 segment. Dijkema et al. (1984) showed that the rat genome contains several alpha-interferon genes, among which at least one, the alphai-interferon gene, is functional; this gene is contained in a 2-5 kb EcoRl restriction fragment 158 M. 0. Islam et al. M R 1 2 3 4 7- 25- 1-1- * Fig. 8. Southern blot analysis showing the loss of rat alphainterferon sequences in the transformed BS sublines. The DNAs were digested with EcoRl and the filter was probed with the rat alpha!-interferon gene. Lanes: M, mouse DNA (BWTG3); R, rat DNA; 1, BS181; 2, BSHOhl; 3, BS181hl; 4, BS181a3; note that the rat 2-5 and 7kb fragments are lost in the transformed sublines. of rat genomic DNA. As illustrated in Fig. 8, we found that this 2-5 kb fragment is lost in the transformed BS140 hybrids of the h- and a-series, which lack rat chromosome 5, and also in the transformed BS181 hybrids of the hand a-series, which possess the deleted rat chromosome 5. Thus, the rat alpha r interferon gene is located in 5q2223. The probe also revealed another rat EcoRl fragment of about 7 kb, which very probably corresponds to another alpha-interferon gene. This fragment was also lost in the transformed hybrid sublines of BS140 and BS181. Therefore, we conclude that the deletion covers at least part of the alpha-interferon locus (possibly the whole locus) and includes the functional alphapinterferon and at least another alpha-interferon gene. Discussion The present work was undertaken to study suppression of the transformed phenotype (anchorage independence) in interspecific somatic cell hybrids (mouse hepatoma X normal rat fibroblasts), which turned out to have the remarkable and extremely favourable property of segregating only a minimum of chromosomes at mitosis. This unusual mitotic stability of the interspecific BS-hybrids enabled us to determine unequivocally the identity of each and every chromosome and to decide easily whether it came from the transformed or the normal parent. This fortunate situation led us to the most significant result of the present study: we established that the anchorage dependence was under control of a gene (group of genes) in rat chromosome 5, or more precisely in 5q22-23. The question arises of whether recovery of many transformed sublines represents independent events. By comparing the chromosome composition of each subline (especially the marker chromosomes) it becomes apparent that there were clear-cut differences in karyotype make-up among the individual sublines of each primary hybrid. By itself this is not necessarily evidence for their independent origin. As is evident from the karyotype data presented, there is a moderate chromosomal evolution in these hybrids, and, although the data provide unmistakable evidence that the sublines are quite distinct from each other, the simplest explanation, in our opinion, is that they have been derived from subpopulations present in the hybrids, which could easily have originated from single deviating cells. This interpretation is supported by the facts that: (1) the deletion of chromosome 5 present in the a- and h-sublines of BS181, morphologically appears to be identical in all of the sublines; and (2) the aand h-sublines of BS140 all carried the same rat-derived l l p + marker chromosome (Table 5). A few hybrids (type II hybrids), which had acquired an excess of mouse hepatoma chromosomes and lost rat chromosomes, retained the transformed properties of the parental hepatoma cells. As discussed (Szpirer & Szpirer, 1980), the lack of suppression in these hybrids is probably due to gene-dosage effects. Indeed, we found (data not shown) that non-deleted rat chromosome 5 is present in some such hybrids; consequently, one can suggest either that the hepatoma chromosome homologous to rat chromosome 5, namely mouse chromosome 4, carries a mutated version of the SAI1 gene, encoding an altered SAI1 product competing with the normal rat SAI1 product, or that other mouse hepatoma chromosomes contribute genes directing the synthesis of proteins antagonizing the rat SAI1 product. Such a possibility might be illustrated by the recently discovered association between oncogene and anti-oncogene (emerogene) products (Whyte et al. 1988). Our conclusion about the SAI1 gene in rat chromo- some 5 must be considered in the light of conclusions from earlier cases of similar work in the mouse. Jonasson et al. (1977) reported that in intraspecific mouse hybrids, malignant X normal, there was a selective pressure both in vivo and in vitro acting against the copies of chromosome 4 derived from the normal parent. They concluded that the locus against which the selective pressure was directed must be 'in the lower part of the upper half of mouse chromosome 4. Again, Evans et al. (1982) confirmed that in similar crosses the suppression of tumorigenicity emanated from chromosome 4 of the normal parent. Several workers have pointed out that the G-banding pattern of rat chromosome 5 is very similar to that of mouse chromosome 4. Gene assignments in the rat determined by other workers and by ourselves (for reviews, see Levan et al. 1986; O'Brien, 1987; Szpirer et al. 1988) have shown that the gene contents of rat chromosome 5 and mouse chromosome 4 exhibit a considerable degree of homology. The similarity in morphology of these two chromosomes is evident (Fig. 9). We have tried to interpret the information in the paper by Jonasson et al. (1977) in terms of the banding nomenclature of Nesbitt & Francke (1973) and have indicated to the right of the figure the approximate limits of the chromosome segment that Jonasson et al. identified as being counterselected against. Assuming the gene homology between mouse chromosome 4 and rat chromosome 5 is reflected by their morphological homology, the deletion found by us in rat chromosome 5 of the nonsuppressed BS181 sublines (a- and h-series) corresponds to part of the segment of mouse chromosome 4 implicated in the study of Jonasson et al. Hence, it seems quite possible that the SAI1 gene in rat chromosome 5 is homologous to the mouse suppressor gene detected by Jonasson et al. (1977). Rat chromosome 5 (and mouse chromosome 4) share gene homology with human chromosomes 1 (the short arm; lp) and 9 (see Searle et al. 1987). Consequently, it is extremely exciting that the presence of a suppressor gene, acting both /// vitro and in vivo, located in the short arm of human chromosome 1 has been indicated in several lines of inquiry, including the following: (1) in somatic cell hybrids between malignant Syrian hamster cells and normal human fibroblasts, anchorage dependence was correlated with the presence of the short arm of human chromosome 1 (Stoler & Bouck, 1985). (2) In intraspecific hybrids between human sarcoma cells and normal human fibroblasts, retention of chromosome 1 was apparently correlated with suppression of the malignant phenotype (Benedict et al. 1984). (3) Deletions and/or rearrangements of the terminal portion of lp (Ip31-lpter) are very common in human neuroblastomas (Brodeur et al. 1981; Gilbert et al. 1982; Kaneko et al. 1985), indicating the presence of an emerogene in this segment. (4) Finally, in a search for loss of heterozygosity in tumour DNA from 15 blood/tumour pairs in patients with the dominantly inherited cancer syndrome multiple endocrine neoplasia type II, Mathew et al. (1987) demonstrated loss of heterozygosity for a probe specific for the short arm of chromosome 1 in seven out of 14 Suppressor gene in rat chromosome 5 159 informative pairs. The chromosomal specificity of the loss was tested on the same filters with probes from five other chromosomes and loss of heterozygosity was observed in no case. Heterozygosity was also conserved for a probe specific for the long arm of chromosome 1. Thus, the SAI1 gene in rat chromosome S might be homologous to an emerogene located in human chromosome 1. The only locus, however, that we could assign to the rat 5q22-23 region was the alpha-interferon locus. Obviously, the presence of this locus in the chromosome region containing the SAI1 gene may be fortuitous and the two loci may just be closely linked. A rough estimate of the size of the deletion gave the approximate value of 10 or 20 megabases; a region of that size might harbour many genes. Still, the possibility remains that the SAI1 gene in fact is an alpha-interferon gene. It is well known that interferons possess growth-inhibiting properties (Taylor-Papadimitriou, 1984). In any case, this finding puts the human chromosome 9 into focus, since the alpha-interferon maps to 9p22-13 in man (Trent et al. 1982). In early reports, evidence from somatic cell hybrids was presented indicating that human chromosome 9 would be involved in tumour suppression (Klinger et al. 1978; Klinger, 1982). We have found that in hybrids between mouse L cells and human fibroblasts, which segregate human chromosomes, the human chromosome 9 is lost preferentially (Wathelet et al. 1988; and our unpublished observations), possibly indicating that hybrids containing human chromosome 9 are at a growth disadvantage. A similar finding has been reported by Ruddle & Creagan (1975). Even stronger indications that human chromosome 9 contains an emerogene can be found by a systematic search for specific chromosome changes in human tumours. A computer printout from the database on chromosome aberrations in human cancer (see Mitelman, 1988) showed that bands 9p22-13 were often involved in deletions or translocations. The overall frequency of 2-4% among all 9029 individual cases present in the registry might not sound so impressive, but in some specific types of malignancies the frequency was quite substantial. Thus, deletions or translocations in bands ft 5q22 5q23 4A4 4C3 RNO5 MMU4 Fig. 9. Diagrams and actual instances of rat chromosome 5 (left) and mouse chromosome 4, showing the morphological similarity between the two chromosomes. On the far left the extent of the deletion in the anchorage-independent sublines of BS181 has been indicated. On the far right we have indicated (in terms of the terminology of Nesbitt & Francke, 1973) the extreme limits for the segment including an emerogene detected by Jonasson et al. (1977) in the mouse. 9p22-13 were found in 70 cases (8-5 %) of acute lymphocytic leukaemias. On the basis of the prevalence of 9p aberrations in this disease, Chilcote et al. (1985) actually suggested that an emerogene must be present in this region. Furthermore, a t(9; 11) translocation involving bands 9p22-13 was very common (13-7%) in acute monocytic leukaemia and in about half of the cases it represented the only aberration detected. Finally, in some solid tumours deletions and translocations in 9p2213 were common (e.g. in more than 50% of astrocytomas). A compilation of the findings is presented in Table 7. Last but not least, homozygous deletions of the alpha- and beta-interferon genes have recently been discovered in human leukaemia cell lines (Diaz et al. 1988). Taken together, these data strongly indicate that an emerogene may be located in the short arm of human chromosome 9. This emerogene may well be homologous to our rat SAI1 gene. Access to the Registry of Chromosome Aberrations in Human Neoplasms was generously provided by Professor Felix Mitelman, Lund. We thank Michele Riviere for technical assistance and Ton Kos, TNO Primate Center, Rijswijk, Holland, for the gift of pPCl. This work was supported by grants from the Swedish Cancer Society, the Erik PhilipSorensen Foundation, the Nilsson-Ehle Foundation and CAN- Table 7. Cases of human neoplastic disease with involvement of chromosome breakage at bands 9p22-13 Number of cases with involvement of9p22-13 Overall frequency *i m / ^ t i fr o i l r"i Cfc dlllOIIg all Ld&eb Disease group Acute non-lymphocytic leukaemia Deletion Translocation 6(1) 1 44 (22) Myeloproliferative disorders Myeiodysplastic syndromes Acute lymphocytic leukaemia Chronic lymphoproliferative disorders 2(2) 41(7) 2 Malignant lymphomas 12 Epithelial neoplasms Mesenchymal neoplasms Melanocytic neoplasms Malignant neurogenic neoplasms 4 1 2 14 5 1 29(6) 9(1) 18 15(2) 4 2 8 Total registered (%) 50 (23) 2-4 6 3(2) 70(13) 11(1) 0-3 0-3 8-5 30 19(2) 5 4 22 1-8 2-9 2-4 2-6 6-9 9-8 Data derived from the Registry of Chromosome Aberrations in Human Cancer (Mitelman, 1988). Numbers in parenthesis refer to cases where an aberration involving 9p22-13 was the only aberration detected in the tumour cells. 160 M. Q. Islam et al. CIRCO (Gothenburg), and the CGER-ASLK, the Belgian Ministere de la Politique Scientifique, and the ASCC-SVTK (Brussels). C. S. and J.-F. D. are senior research associate and research assistant, respectively, of the National Fund for Scientific Research (Belgium). References BENEDICT, W. F., WEISSMAN, B. E., MARK, C. & STANBRIDGE, E. J. (1984). Tumorigenicity of human HT1080 fibrosarcoma X normal fibroblast hybrids: Chromosome dosage dependency. Cancer Res. 44, 3471-3479. BRODEUR, G. M., GREEN, A. A., HAYES, F. A., WILLIAMS, K. J., WILLIAMS, D. L. & TSIATIS, A. A. (1981). Cytogenetic features of human neuroblastomas and cell lines. Cancer Res. 41, 4678-4686. CAVENEE, W. K., DRYJA, T. P., PHILIPS, R. A., BENEDICT, W. F., GODBOUT, R., GALLIE, B. L., MURPHEE, A. L., STRONG, L. C. & WHITE, R. L. (1983). Expression of recessive alleles by chromosomal mechanisms in retinoblastoma. Nature, Loud. 305, 779-784. CHILCOTE, R. R., BROWN, E. & ROWLEY, J. D. (1985). Lymphoblastic leukemia with lymphomatous features associated with abnormalities of the short arm of chromosome 9. A'. Engl. J. Med. 313, 286-291. COMMITTEE FOR A STANDARDIZED KARYOTYPE OF RATTUS NORVEGICUS. (1973). Standard karyotype of the Norway rat, Rattus norvegicus. Cytogenet. Cell Genet. 12, 199-205. COMMITTEE ON STANDARDIZED GENETIC NOMENCLATURE FOR MICE. (1972). Standard karyotype of the mouse, Mus musadus. J. Hered. 63, 69-72. CRAIG, R. W., GADI, 1. K. & SAGER, R. (1988). Genetic analysis of tumorigenesis. XXXI: Retention of short arm of chromosome 3 in suppressed CHEF cell hybrids containing c-Ha-ras (EJ) gene. Somat. Cell molec. Genet. 14, 41-53. DIAZ, M. O., ZIEMIN, S., L E BEAU, M. M., PITHA, P., SMITH, S. D., CHILCOTE, R. R. & ROWLEY, J. (1988). Homozygous deletion of the a- and ^pinterferon genes in human leukemia and derived cell lines. Proc. natn. Acad. Sci. U.S.A. 85, 5259-5263. DlJKEMA, R., POUWEL, P., DE REUS, A. & SCHELLEKENS, H. (1984). Structure and expression in Escherichia coli of a cloned interferono-gene. Nucl. Acids Res. 12, 1227-1242. DYSON, P. J., COOK, P. R., SEARLE, S. & WYKE, J. A. (1985). The chromatin structure of Rous sarcoma provirus is changed by factors that act in trans in cell hybrids. EMBOJ. 4, 413-420. EVANS, E. P., BURTENSHAW, M. D., BROWN, B. B., HENNION, R. & HARRIS, H. (1982). The analysis of malignancy by cell fusion. IX. Re-examination and clarification of the cytogenetic problem. J. Cell Sci. 56, 113-130. FEINBERG, A. P. & VOGELSTEIN, B. (1984). A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analvt. Btochein. 137, 266—267. tumorigenicity in intraspecies hybrids of normal diploid X malignant cells. Cytogenet. Cell Genet. 41, 65-70. KANEKO, Y., TSUCHIDA, Y., MASEKI, N., TAKASAKI, N., SAKURAI, M. & SAITO, S. (1985). Chromosome findings in human neuroblastomas Xenografted in nude mice, jfpn J. Cancer Res. 76, 359-364. KLEIN, G. (1987). The approaching era of the tumor suppressor genes. Science 238, 1539-1550. KLINGER, H. P. (1980). Suppression of tumorigenicity in somatic cell hybrids. I. Suppression and reexpression of tumorigeneicity in diploid human x D98AH2 hybrids and independent segregation of tumorigenicity from other cell phenotypes. Cvtoqenet. Cell Genet. 27, 254-266. KLINGER, H. P. (1982). Suppression of tumorigenicity. Cvtogenet. Cell Genet. 32, 68-84. KLINGER, H. P., BAIM, A. S., EUN, C. K., SHOWS, T. B. & RUDDLE, F. H. (1978). Human chromosomes which affect tumorigenicity in hybrids of diploid human with heteroploid human or rodent cells. Cytogenet. Cell Genet. 22, 245-249. KLINGER, H. P. & KAELBLING, M. (1986). Suppression of tumorigenicity in somatic cell hybrids. IV. Chromosomes of normal human cells associated with suppression of tumorigenicity in hybrids with D98AH2 carcinoma cells. Cvtogenet. Cell Genet. 42, 225-235. KNUDSON, A. G. JR (1985). Hereditary cancer, oncogenes, and antioncogenes. Cancer Res. 45, 1437-1443. LEVAN, G. (1974). Nomenclature for G-bands in rat chromosomes. Hereditas 77, 37-52. LEVAN, G., SZPIRER, J., SZPIRER, C. & YOSHIDA, M. C. (1986). Present status of chromosome localization of rat genes. Rat News Utter 17, 3-8. LITTLEFIELD, J. W. (1964). Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710. MACPHERSON, I. & MONTAGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291-294. MARSHALL, C. J. (1980). Suppression of the transformed phenotype with retention of the viral src gene in cell hybrids between Rous sarcoma virus-induced rat cells and untransformed mouse cells. Expl Cell Res. 127, 373-384. MARSHALL, C. J. & DAVE, H. (1978). Suppression of the transformed phenotype in somatic cell hybrids. J. Cell Sci. 33, 171-190. MARSHALL, C. J. & SAGER, R. (1981). Genetic analysis of tumorigenesis: IX. Suppression of anchorage independence in hybrids between transformed hamster cell lines. Somat. Cell Genet. 7, 713-723. MARTINSSON, T., TENNING, P., LUNDH, L. & LEVAN, G. (1982). Methotrexate resistance and double minutes in a cell line from the SEWA mouse ascites tumor. Hereditas 97, 123-137. MATHEW, C. G. P., SMITH, B. A., THORPE, K., WONG, Z., ROYLE, T. (1969). Suppression of malignancy by cell fusion. Nature, Loud. 223, 363-368. ISLAM, M. Q. & LEVAN, G. (1987). A new fixation procedure for improved quality G-bands in routine cytogenetic work. Hereditas 107, 127-130. JONASSON, J., POVEY, S. & HARRIS, H. (1977). The analysis of malignancy by cell fusion. VII. Cytogenetic analysis of hybrids between malignant and diploid cells and of tumours derived from them. J Cell Sci. 24, 217-254. N. J., JEFFREYS, A. J. & PONDER, B. A. (1987). Deletion of genes on chromosome 1 in endocrine neoplasia. Nature, Land. 328, 524-526. MITELMAN, F. (1988). Catalog of Chromosome Aberrations in Cancer, 3rd edn. New York: Alan R. Liss. NESBITT, M. N. & FRANCKE, U. (1973). A system of nomenclature for band patterns of mouse chromosomes. Chromosoma 41, 145-158. O'BRIEN, S. J. (1987). Genetic Maps 19S7. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. RUDDLE, F. H. & CREAGAN, R. P. (1975). Parasexual approaches to the genetics of man. A. Rev. Genet. 9, 407-486. SAGER, R. (1985). Genetic suppression of tumor formation. Adv. Cancer Res. 44, 43-68. SAGER, R. (1986). Genetic suppression of tumor formation: A new frontier in cancer research. Cancer Res. 46, 1573-1580. SAGER, R. & KOVAC, P. E. (1978). Genetic analysis of tumorigenesis: I. Expression of tumor-forming ability in hamster hybrid cell lines. Somat. Cell Genet. 4, 375-392. KAELBLING, M. & KLINGER, H. P. (1986). Suppression of SAXON, P. J., SRIVATSAN, E. S. & STANBRIDGE, E. J. (1986). GILBERT, F., BALABAN, G., MOORHEAD, P., BIANCHI, D. & SCHLESINGER, H. (1982). Abnormalities of chromosome lp in human neuroblastoma tumors and cell lines. Cancer Genet. Cytogenet. 7, 33-42. HARRIS, H. (1985). Suppression of malignancy in hybrid cells: the mechanism. 7. Cell Sci. 79, 83-94. HARRIS, H. (1986). The genetic analysis of malignancy. J. Cell Sci. Suppl. 4, 431-444. HARRIS, H., MILLER, O. J., KLEIN, G., WORST, P. & TACHIBANA, tumorigenicity in somatic cell hybrids. III. Cosegregation of human chromosome 11 of a normal cell and suppression of Introduction of human chromosome 11 via microcell transfer controls tumorigenic expression of HeLa cells. EMBO J. 5, Suppressor gene in rat chromosome 5 161 3461-3466. SEARLE, A. G., PETERS, J., LYON, M. F., EVANS, E. P., EDWARDS, J. H. & BUCKLE, V. J. (1987). Chromosome maps of man and mouse, 111. Genomics 1, 3-18. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 98, 503-518. SRIVATSAN, E. S., BENEDICT, W. F. & STANBRIDGE, E. J. (1986). Implication of chromosome 11 in the suppression of neoplastic expression in human cell hybrids. Cancer Res. 46, 6174-6179. STANBRIDGE, E. J. (1976). Suppression of malignancy in human cells. Nature, Land. 260, 17-20. STANBRIDGE, E. (1985). A case for human tumor-suppressor genes. ' BioEssays 3,252-255. STANBRIDGE, E. J., DER, C. J., DOERSEN, C.-J., NISHIMI, R. Y., PEEHL, D. M., WEISSMAN, B. E. & WILKINSON, J. E. (1982). Human cell hybrids: Analysis of transformation and tumorigenicitv. Science 215, 252-259. STANBRIDGE, E. J., FLANDERMEYER, R. R., DANIELS, D. W. & NELSON-REES, W. A. (1981). Specific chromosome loss associated with the expression of tumorigenicitv in human cell hybrids. Somat. Cell Genet. 7, 699-712. STANBRIDGE, E. J. & WILKINSON, J. (1978). Analysis of malignancy in human cells: Malignant and transformed phenotypes are under separate genetic control. Proc. natn. Acad. Sci. U.S.A. 75, 1466-1469. STOLER, A. & BOUCK, N. (1985). Identification of a single chromosome in the normal human genome essential for suppression of hamster cell transformation. Pmc. natn. Acad. Sci. U.S.A. 82, 570-574. STRAUS, D. S., JONASSON, J. & HARRIS, H. (1976). Growth in vitro of tumour cell X fibroblast hybrids in which malignancy is suppressed. J. Cell Sci. 25, 73-86. SZPIRER, J. & SZPIRER, C. (1979). Production of serum proteins in normal diploid fibroblast-hepatoma cell hybrids and in A9—normal 162 M. O. Islam et al. liver cell hybrids. J. Cell Sci. 35, 267-279. SZPIRER, C. & SZPIRER, J. (1980). Suppression of the transformed phenotype of hepatoma cells after hybridization with normal diploid"fibroblasts. Expl Cell Res. 125, 305-312. SZPIRER, C , SZPIRER, J., ISLAM, M. Q. & LEVAN, G. (1988). The rat gene map. Cttrr. Top. microbiol. Iniimin. 137, 33-38. TAYLOR-PAPADIMITRIOU, J. (1984). Effects of interferons on cell growth and function. In Inteiferon, vol. 1: General and Applied Aspects (ed. A. Billiau), pp. 136-166. Amsterdam: Elsevier. TODARO, G. (1988). In Theories of Carcinogenesis (ed. O. H. Iversen), pp. 61-80. Washington, DC: Hemisphere. TRENT, J. M., OLSON, S. & LAWN, R. M. (1982). Chromosomal localization of human leukocyte, fibroblast, and immune interferon genes by means of in situ hybridization. Proc. natn. Acad. Sci. U.S.A. 79, 7809-7813. WATHELET, M., SZPIRER, J., NOLS, C , KLAUS, I., DE WIT, L., ISLAM, M. Q., LEVAN, G., HORISBERGER, M., CONTENT, J., SZPIRER, C. & HUEZ, G. (1988). Cloning and chromosomal location of human genes inducible by type I interferon. Somat. Cell molec. Genet. 14, 415-426. WHYTE, P., BUCHKOVICH, K. J., HOROWITZ, J. M., FRIEND, S. H., RAYBUCK, M., WEINBERG, R. A. & HARMOW, E. (1988). Association between an oncogene and anti-oncogene: the adenovirus El A proteins bind to the retinoblastoma gene product. Nature, Loud. 334, 124-129. WIENER, F., KLEIN, G. & HARRIS, H. (1971). The analysis of malignancy by cell fusion. III. Hybrids between diploid fibroblasts and other tumour cells. J. Cell Sci. 8, 681-692. WIENER, F., KLEIN, G. & HARRIS, H. (1974). The analysis of malignancy by cell fusion. V. Further evidence of the ability of normal diploid cells to suppress malignancy. J. Cell Sci. 15, 177-183. (Received 2 August I98S -Accepted 7 October 1988)