endothelial cell tumors develop in transgenic mice carrying polyoma virus middle t oncogene

Cell, Vol. 51, 529-538, November 20, 1997, Copyright 0 1987 by Cell Press

Endothelial Cell Tumors Develop in Transgenic Mice Carrying Polyoma Virus Middle T Oncogene

Victoria L. Bautch,’ Sachiko Toda,’ John A. Hassell,t and Douglas Hanahan’ l Cold Spring Harbor Laboratory Cold Spring Harbor, New York 11724 t Department of Microbiology and Immunology McGill University Montreal, Quebec Canada

Summary

Inoculation of newborn mice with the murine polyoma (Py) virus leads to tumor formation in a wide range of tissues. In order to investigate viral oncogenesis, we generated transgenic mice carrying eitherthe Py large T antigen (LT) gene or the Py middle T antigen (MT) gene linked to Py early region regulatory sequences. While Py LT mice exhibit no phenotype, Py MT mice develop multifocal tumors of the vascular endotheli- urn. These hemangiomas are lethal to the animals and can be passaged in vivo. Transgene RNAs and protein are present in both hemangiomas and the testes of these mice, and the Py middle T protein in both tissues is complexed to a cellular tyrosine kinase. The expression of complexed middle T protein in both tumori- genie endothelial cells and unperturbed testes implies that endothelial cells may be particularly susceptible to the action of the middle T oncogene. These observations indicate that Py middle T disrupts the normal strict controls on vascular growth, and suggest that Py MT transgenic mice will provide a model for studying the control of angiogenesis.

Introduction

Tumorigenesis is a complex multistep process involving unregulated proliferation, evasion of immune surveil- lance, vascularization, development of invasive potential, and metastatic spread. Indirect evidence of tumor progression from the epidemiology of human cancer is supported by the definition of oncogene classes that cooperate to transform primary cells in culture (Rassoulzadegan et al., 1982; Land et al., 1983; Ruley, 1983; reviews, Farber and Cameron, 1980; Bishop, 1985, 1987; Klein and Klein, 1985; Weinberg, 1985; Knudson, 1986). The introduction of oncogenes into transgenic mice provides a means for studying the parameters governing tumorigenesis in vivo at the molecular level. Recent results using this approach have indicated first, that single oncogenes can cause tumors, but often with a latency that suggests a require- ment for additional genetic or epigenetic alterations (Stewart et al., 1984; Hanahan, 1985; Adams et al., 1985; reviews, Palmiter and Brinster, 1986; Hanahan, 1986); second, that members of different oncogene classes can cooperate in vivo to accelerate tumorigenesis (Sinn et al.,

1987); and third, that some oncogenes exhibit a cell specificity in their action in vivo (Adams et al., 1985; Leder et al., 1986; Ruther et al., 1987).

The viral oncogenes of papovaviruses are particularly well-suited to establishing correlations between transformation in culture and tumorigenesis in vivo. The transforming genes of both S/40 and polyoma (Py) have been extensively characterized in cultured cells, and they can be tumorigenic in mice (Hargis and Maikiel, 1979; Abram- czuk et al., 1984; reviews, Tooze, 1981; Eddy, 1982). The discovery that the SV40 early region encoding SV40 T antigen produces tumors of the choroid plexus in transgenic mice (Brinster et al., 1984; Palmiter et al., 1985) has been followed by demonstrations of tumorigenicity in diverse cell types by linking the SV40 T antigen gene to heterolo- gous promoters (Hanahan, 1985; Ornitz et al., 1985; Ma- hon et al., 1987). The analogous early regions of the human DNA tumor viruses JC and BK cause perturbations in specific tissues of transgenic mice, and these tissues correspond to the sites of virus isolation in humans (Small et al., 1986). The BPV genome also recapitulates its bovine tumor spectrum by causing skin tumors in transgenic mice (Lacey et al., 1986). Dissection of the oncogenic process in vivo, however, is hampered in these systems because S/40 large T antigen carries the full transforming potential of SV40 virus, and the transforming genes of JC, BK, and BPV are not well characterized.

We chose to investigate the effects of the Py virus early region genes in transgenic mice. The natural host of Py virus is the mouse, and infection of adults by normal routes is asymptomatic. Inoculation of newborn mice with high titers of virus, however, results in tumor formation in a broad range of tissues, with tumors of the parotid and other salivary glands being the most prevalent (reviews, Gross, 1970; Eddy, 1982). The early region of Py, required for both lytic functions and transformation, encodes three proteins that arise by differential splicing of a primary transcript: large T antigen is a 100 kd nuclear protein required for viral replication, middle T antigen is a 56 kd membrane protein with transforming activity, and small T antigen is a 22 kd protein of undefined function (Tooze, 1981). Most significantly, large T is capable of immortalizing primary cells to continuous growth in culture, while middle T con- fers the transformed phenotype to immortalized cells. These two oncogenes can cooperate to transform primary cells in culture (Treisman et al., 1981b; Rassoulzadegan et al., 1982). Analysis of the effects of the Py virus early region in transgenic mice, therefore, provides a way to study the separate and concerted action of viral oncogenes in their natural host.

In this study, transgenic mice carrying replication-defective Py virus early region regulatory sequences linked to cDNAs encoding either Py large T or Py middle T have been constructed. Large T transgenics exhibit no phenotype, whereas middle T transgenics develop hemangiomas, a tumor of endothelial cell origin.

Cell 530

Figure 1. Py LT and Py MT Transgenic DNAs

The upper diagram shows the prototype (replication-defective) Py DNA with bp 37-60 deleted and replaced by an Xhol linker, and a deletion in the late region coding sequences. Dashed line, vector DNA; open line, late region DNA; dotted area, Py enhancer; parentheses, deleted sequences. The lower diagrams show the cDNAs for LT and MT, which replaced the wild-type early coding sequences in the microinjected constructs. The dashed lines represent introns (not present in constructs) and the filled line denotes the protein coding sequences. Py nucleotides are numbered according to Soeda et al. (1960).

Results

Py DNAs and Transgenic Lineages The Py DNAs injected into mouse embryos are shown in Figure 1. The DNAs are composed of a replication- defective Py early regulatory region linked to cDNAs encoding either large T (Py LT) or middle T (Py MT). Both DNAs were shown to produce wild-type levels of functional proteins: Py LT can complement a Py origin to support replication in tram in a cotransfection assay, and Py MT can transform NIH3T3 cells (Muller et al., 1983; J. Has- sell, unpublished results). These DNAs were injected into fertilized eggs after linearization with Sall, and the resulting mice were screened at two weeks of age for the presence of the transgenes. Two of 16 live pups injected as embryos with Py LT were transgenic, and two of 25 live pups injected with Py MT were transgenic. All Py transgenes were integrated into chromosomal DNA as judged by Southern blot analyses of both high molecular weight DNA and low molecular weight DNA (data not shown).

The fate of the transgenic founders is documented in Ta- ble 1. Both mice carrying Py LT transmitted the transgene. The founder of PyLT-1 died at ten months with no visible or microscopic abnormalities; other mice of this lineage, however, are alive and apparently well at one year of age. The founder of PyLT-2 is alive at 15 months of age, and other members of this lineage appear healthy. These results indicate that the presence of Py large T in the genome does not produce any obvious effects on the mice.

In contrast, the mice harboring Py MT develop hemangiomas, which are tumors of the vascular endothelium. The founder of lineages PyMT-1A and PyMT-1B (#138) was found moribund at 2.5 months of age and sacrificed. The animal suffered from anemia and was diagnosed as having multiple hemangiomas of the liver, lung, mesen- tery, and subcutaneous tissue. A similar pathology arose in an independently derived transgenic mouse, PyMT-2 (#137), who succumbed at 6.5 months of age. Histopatho- logical analysis of multiple tissues of both mice revealed no pathology other than hemangiomas, and both mice

carried hemangiomas at multiple sites. The documenta- tion of this pathology in two founder mice argues that the phenotype is not due to a specific insertion event into chromosomal DNA.

The two Py MT lineages described below (PyMT-1A and PyMT-1B) derive from founder mouse #138. One progeny of #138 appeared to carry fewer copies of the transgene that its siblings, and this cq:y number difference bred true in subsequent generations. Different lineages have been provisionally designated based on the assumption that the founder carried two independent integration sites of Py MT DNA, although it is formally possible that some transgene copies were lost from a single integration site to generate the low copy number animal. Mice of both sub- lines develop fatal hemangiomas. The independent trans- genie mouse #137 (PyMT-2) did not transmit the Py MT transgene to any of his progeny (see Table l), and may have been mosaic for the transgene.

The Py MT-1A Lineage The Py MT-1A lineage was expanded for further analysis, and a partial pedigree of this lineage is shown in Figure 2. Transgenic mice were mated to nontransgenic mice of the inbred strains C57BLEJ (B6) or DBARJ (D2), and to nontransgenic B6D2 Fl mice. These results show that the transgene was transmitted to approximately 50% of each generation, as expected for heterozygous animals. The most striking observation is that all mice of the first and second generations (Gl and G2, lines 2 and 3 of the pedigree) carrying the Py MT transgene died or were ill and sacrificed. All sacrificed mice carried hemangiomas upon autopsy. These results indicate that the effects of the transgene are dominant and exhibit 100% penetrance. These transgenic mice are of mixed genetic background because the founder was an F2 hybrid between the C57BL16J and DBA/2J strains; thus the complete penetrance of the phenotype suggests that genetic background does not affect the qualitative expression of the Py MT phenotype. The average age of death was 13.9 weeks. It is notable, however, that the average age of death has increased in each succeeding generation, so that Gl was 8.75 weeks, G2 was 11.5 weeks, and G3 was 23.3 weeks. This effect may be due to changes in genetic background, because most mice were backcrossed to DBARJ mice. Al- ternatively, our breeding methods may impose a selection for long-lived animals because these mice live to produce more offspring.

The hemangiomas are blood-filled cysts in a range of sizes from barely visible to as large as 1.5 cm3 (Figure 3A). Most mice exhibited multiple hemangiomas, with the most common sites being the liver, lungs, and subcutaneous tissue, all well-vascularized organs. The tumors were often found clustered in a single organ with a few tumors elsewhere, rather than evenly dispersed along the vas- culature. For example, the mouse whose lungs carry multiple tumors in Figure 3A had only 5-10 visible tumors in the liver and one in the spleen. Histological examination of the tumors indicates that they are primarily a two- dimensional proliferation of the vascular endothelial cells,

Hemangiomas in PyMT Transgenic Mice 531

Table 1. Polyoma Transgenic Animals and Lineages

Construct Founder Agea Lineage Copy No.~ Pathology

PyLT #I40 10 months (D) PyLT-1 2-10 none detected #I42 15 months (A) PyLT-2 30-50 none detected

PyMT #138 2.5 months (D) PyMT-1 A 30-50 hemangiomas - PyMT-10 2-10 hemangiomas

#137 8.5 months (D) PyMT-2’ ND hemangiomas

a Current age of founder or age at death; (A) alive, (D) dead. b No lineage was established from the PyMT-2 mouse because he did not transmit the transgene to 159 tested progeny. c Estimates of haploid copy number of the transgene are derived from reconstructions with plasmid DNA and Southern blot analysis.

such that the cylindrical vessel develops into a distended sac that becomes filled with blood components (Figures 3B, 3C and 30). Larger hemangiomas, however, had some connected endothelial cells resembling sprouts projecting into the lumen (Figure 3, arrows), and networks of endothelial cells were occasionally seen (Figure 3C). Most mice appeared anemic and had enlarged spleens, presumably a result of increased hematopoiesis stimulated by the anemia. Some animals died from internal hemor- rhage because of rupture of a hemangioma, while others sustained no visible rupture and apparently died from anemia.

To better characterize the tumorigenic potential of these hemangiomas, they were transplanted into histocompati- ble 8602 Fl hosts. The results, documented in Table 2, indicate that tumor tissue will grow when implanted into subcutaneous sites of nontransgenic Fl mice. The passaged tumors were lethal to the hosts, and autopsies of these animals revealed that each had developed a subcutaneous hemangioma and was suffering from anemia. No metastases were observed. Both the MT-1A tumors and

I421 1179

Figure 2. Partial Pedigree of MT-1A Lineage

This pedigree was compiled from birth and death records. Transgenic animals were identified as described (see Experimental Procedures). Filled symbols, MT transgenic mice; open symbots, nontransgenic mice; squares, males; circles, females. Diagonals: solid diagonal, found dead; solid diagonal + S, sacrificed; dashed diagonal, missing and presumed dead. Each series of symbols connected from above with a horizontal line is one litter (the centered number is the litter number), and symbols connected from below with a horizontal line denote matings. The numbers directly below a symbol indicate lifespan of the animal in weeks. The animal with the hatched symbol and the asterisk carried a lower copy number of the transgene than siblings, and was used to expand an independent lineage (MT-1B).

tumors excised from the independently derived MT-2 mouse were transplantable, albeit with different latencies, indicating that this property is inherent to Py middle T induced hemangiomas. These results show that the middle T induced hemangiomas are tumorigenic at subcutaneous sites in nontransgenic hosts and can invade intact tissue. The confined nature of the host-derived tumors suggests that the metastatic potential of the tumor cells is low.

Expression of Py Transgene RNA The specificity of the pathology observed in the Py MT transgenic mice suggested that there is also specificity in the expression of the Py middle T gene and/or in the action of the Py middle T protein. To address these possibili- ties, we determined the in vivo expression pattern of the genes. An RNA protection assay was employed to identify transgene RNA. A riboprobe construct containing a portion of the Py genome (PxBP64, Figure 4, lower panel) was transcribed in vitro with SP6 polymerase to generate an antisense RNA probe (Melton et al., 1964). The probe fragment protected by large T RNA should be shorter than that protected by the middle T and/or small T RNA because this probe covers the splice donor site for the Py large T RNA (nucleotide 409). The large T RNA should protect 257 bases, while the middle T RNA should protect 332 bases of the probe if initiation occurs at the major sites of early region transcription (see Figure 4, lower panel).

Total RNAs were isolated from 12 tissues of a healthy three month old MT-1A mouse that had a single subcutaneous hemangioma upon autopsy. The RNAs were hybridized to an excess of the 32P-labeled antisense RNA and digested with RNAases. The results (Figure 4A) show that transcripts complementary to the probe were present only in the testes. A similar survey of tissues from another mouse of the MT-1A lineage and of six tissues from a mouse of the LT-2 lineage yielded the same pattern of expression (data not shown). The major protected fragment derived from the MT-1A testes RNA, however, was larger (525 bases) than the fragment protected in control RNA (332 bases), indicating that transcription initiated upstream of the major early start sites.

Testicular RNAs from animals of several different lineages were analyzed (Figure 48). These results show that transcripts complementary to the transgenes are present in testes of both Py LT lineages and both Py MT lineages. These data and analysis of 11 additional transgenic testic-

Cell 532

Figure 3. Pathology of MT-1A Hemangiomas

(A) 3x magnification of intact lungs from a DBARJ mouse (left) and a PyMT-IA transgenic mouse (right). Note the mulhple hemangiomas of various sizes in the PyMT lung (mouse 277112). (B) 57x magnification of a tissue section from the lung shown in (A). A single hemangioma on the periphery of the lung contains blood ceils (mouse 277112). (C) 230x magnification of a mesenteric PyMT-IA hemangioma. The blood cells (lower half) are in a lumen lined with endothelial cells. The arrow points to a sprout-like projection of endothelial cells into the lumen. Note also the network of endothelial cells in the upper part of the photograph (mouse #136). (D) 575x magnification of the PyMT mesenteric hemangioma shown in (C). The arrow points to a projection of endothelial cells into the blood-filled lumen (mouse #136). All tissue sectrons were stained with hematoxylin and eosin.

Table 2. Subcutaneous In Vivo Passage of MT Transgenic Endothelial Tumors In Fl Hosts

Donor

Genotype Mouse Primary Tumor Site

Host

Tumor Frequencya Llfespan Passageb

MT-1A #229/6 subcutaneous 314 4-6 weeks 1 MT-1 A #161 subcutaneous 2/4 8-9 weeks 1 MT-2 #I37 subcutaneous l/l aliveC 1

lung 212 16 week& 1 MT-1A 22916A subcutaneous 213 5-10 weeks 2 MT-1A 229168 subcutaneous 2/3 6-10 weeks 2 - a Number of animals that developed transplanted tumors/number of animals injected with tumor material. b Passage 1 is from transgenic animal to Fl animal. Passage 2 is from one Fi animal to a second Fl animal. c This animal is alive (35 weeks) with a palpable tumor. d Of two animals receiving lung material, one died at 16 weeks and one is alive (35 weeks) with a palpable tumor.

ular RNAs (data not shown) demonstrate that transgene site mapped by Fenton and Basilica (1982). An additional RNAs from all testes initiate upstream of the major sites. protected fragment larger than the major band in testes The 5’ end of the major fragment protected by testicular (Figure 4A, lane 4) probably represents a hybridization ar- RNAs in all lineages maps near several minor early start tifact because the same longer protected fragment was sites (Kamen et al., 1982; see Figure 4, lower panel) and seen in RNAs from both Py MT and Py LT testes, while may coincide with a major late-early transcription start bona fide transcripts were of different lengths (Figure 48).


- 622

MT*- - 527

- 404

MT - - 309

MT*

MT

LT - - 242

LT

I23456769101112131415 I234567 123456

622

- 242

PxBP 64

37 XhoI 60 152 Cl01 154

Figure 4. RNA Analysis of Transgenic Tissues

Total RNAs were isolated from mouse tissues and used in an RNA protection assay. The products were electrophoresed in 8 M urea, 5% polyacrylamide gels. (A) Tissue survey of RNAs from several tissues of one MT-1A animal. Lane 1, NIH 3T3 cells; lane 2, liver; lane 3, spleen; lane 4, testes; lane 5, kidney; lane 6, lung; lane 7, brain; lane 8. pancreas; lane 9, intestine; lane 10, heart; lane 11, thymus; lane 12, salivary glands; lane 13, muscle; lane 14, no sample; lane 15, PyT54 cells (a hamster cell line transformed by Py). (B) RNAs from testes of several different lineages. Lane 1, PyT54 cells: lane 2, mock reaction; lane 3, MT-1A testes; lane 4, MT-1B testes; lane 5, LT-2 testes; lane 6, LT-1 testes; lane 7, Fl testes (nontransgenic). (C) RNAs from different MT-IA hemangiomas. Lane 1, tumor 392/g; lane 2, tumor 369/6; lane 3, tumor 181; lane 4, mock reaction; lane 5, PyT54 cells; lane 6, 244/5 testes. Left margins show fragments corresponding to protection by transcripts. LT, predicted LT transcript; MT, predicted MT transcript; LT: transgenic LT transcript; MT: transgenic MT transcript. The protected fragments in PyT54 cells are trimmed by several nucleotides because the RNAs are not colinear with the probe (see Clal linker insertion on lower map); control experiments with a colinear probe (data not shown) demonstrate that PyT54 RNAs initiate at the major early site. Right margins show size markers, pBR322 digested with Hpall. Exposure times were four days with an intensifying screen. The upper diagram shows the Py genome from nucleotide 4632 to nucleotide 1016. Initiation sites for early transcription (right pointing elevated arrows) and late transcription (left pointing elevated arrows) were mapped by Kamen et al. (1962). Early transcript splice junctions (dotted lines) are according to Treisman et al. (196la). Dark lines, coding sequences; open lines, noncoding sequences, waved arrows, extensions of the RNAs beyond the map coordinates, L, large T antigen RNA; M, middle T antigen RNA; S, small T antigen RNA. Stippled box below shows the BamHI-Pstl fragment cloned into SP64. The lower diagram shows the Py sequences as modified in the constructs used for microinjection. The elevated arrows indicate the ends of the protected fragments in transgenic tissues as mapped by this assay, and parentheses indicate deletions in the DNA. The inserted linkers are indicated below the deletions.

Protected fragments shorter than the major band probably represent RNA degradation products because RNAs from different Py MT testes contained varying amounts of these fragments relative to the major fragment (for example, compare Figure 4A, lane 4 with Figure 48, lane 3). These

minor bands, however, may represent additional initiation sites used with less frequency. The resolution of the RNA protection assay does not allow assignment of the precise 5’ end of the transcripts.

RNAs from several MT-1A hemangiomas were assayed

Cell 534

for the presence of middle T transcripts. The tumor- derived RNAs contained transcripts complementary to the probe (Figure 4C). RNA from one tumor protected a fragment of the same size as testes RNA, whereas two other preparations protected a fragment of the size seen in control RNA (Figure 4C, lanes 2 and 3), indicating initiation at the major site for early transcription. These results indicate that two different transcription initiation sites can be used in these transgenes, and that in tumors either site is utilized.

Expression of Py Middle T Protein Because testicular RNA expression does not appear to lead to any overt pathology or dysfunction, we inves- tigated the expression of middle T protein using an in vitro kinase assay (Lipsich et al., 1983). A fraction of the middle T protein in cultured cells is found complexed to a cellular tyrosine kinase such as C-WC or c-yes, and this complex is thought to be necessary but not sufficient for transformation (Courtneidge and Smith, 1983; Bolen et al., 1984; Kornbluth et al., 1987). The kinase assay, which involves immunoprecipitation and subsequent incubation of the pellet with Y[~~P]-ATP, detects complexed middle T protein because only this species of middle T becomes phos- phorylated in vitro. The complexed cellular kinase undergoes autophosphorylation in this assay.

Several tissues from two mice of the MT-1A lineage were analyzed in this manner, and the results of one assay are shown in Figure 5. The control lysate was immunoprecipi- tated with both C-UC and MT monoclonal antibodies (Fig- ure 5, PyT54). lmmunoprecipitation of tissue lysates with the MT monoclonal antibody demonstrates that kinase reactive middle T protein is detectable in both the tumor tissue and the testes of MT-IA animals. There was no detectable signal in any other tissue, with the exception of lung. The weak signal from lung tissue probably resulted from tumorigenic endothelial cell expression because this animal carried several small hemangiomas in the lung. A higher molecular weight protein coprecipitates with middle T in testes extracts and may be c-src. Although the cellular tyrosine kinase complexed with middle T has not been identified, independent immunoprecipitations of the same tissue lysates with the C-WC monoclonal antibody show that all tested tissues contain C-WC (data not shown). Thus the presence of a complex between the middle T protein and a cellular tyrosine kinase is correlated with pathology of the vascular endothelium, yet it is not as- sociated with any dysplasia in the testes. The assay of middle T expression in preneoplastic endothelial cells will require in situ techniques because the kinase assay, while sensitive, is not likely to detect expression in a cell type that is a small percentage of the cells in each organ.

Discussion

Expression of the Py middle T oncogene linked to its own promoter in transgenic mice causes tumors of a specific cell type, the vascular endothelial cells. The specificity of tumor formation in Py MT transgenic mice differs from the broad tumor spectrum generated by infection of newborn

Figure 5. In Vitro Kinase Assay of Transgenic Tissues

Mouse organs from a single MT-1A animal were homogenized and subjected lo immunoprecipitation as described. The pellets were reacted with @*P]-ATP and the products electrophoresed in an SDS/7.5% polyacrylamide gel. The gel was heated in 1 N KOH prior to drying. Symbols: -, no primary antibody; src+, monoclonal antibody 327 to src; MT+, monoclonal antibody 615 to Py MT antigen. Lanes 1-3, PyT54 cells; lanes 4-5, liver; lanes 6-7, spleen; lanes 6-9, kidney; lanes 10-11, testes; lanes 12-13, lung; lanes 14-15, tumor; lanes 16-17, Fl testes (nontransgenic). Left margin shows migration of C-WC and MT proteins In this gel system, and size markers are shown in the right margin.

mice with intact Py virus. Hemangiomas develop with some frequency in infected mice, but tumors of the salivary glands, thymus, mammary glands, and bone are far more prevalent (Gross, 1970; Eddy, 1982). The restricted specificity of tumors in the transgenic mice indicates that the Py middle T gene alone is not sufficient to recapitulate the tumor spectrum of the virus. Among the missing components that may affect tumorigenesis in nonendothelial tissues are expression of the other early region proteins (large T and small T), viral replication, and expression of late region genes. The fact that tumorigenesis by Py virus is a complex process is illustrated by the recent finding that different strains of Py virus differ in both oncogenic potential and tumor spectrum on the same genetic background, and these differences do not map to a defined portion of the Py genome (Dawe et al., 1987; Freund et al., 1987).

Expression of the Transgenes Expression analysis of the Py MT transgene suggests that specificity of both expression and protein action contrib- ute to the restricted tumor pattern. Detectable levels of transgene RNA and protein were found only in the tumors and the testes of these animals. Lineages of mice carrying Py large T linked to the Py promoter also showed expression only in the testes, indicating that this specificity of expression is not likely to result from integration site position effects. Our analysis would probably not detect activity of the Py promoter in rare cell types or in cell types that are dispersed in different organs. It is surprising, however, that activity of the Py early promoter is not detectable in additional cell types in vivo, given that histological analy-


ses of Py virus-induced tumors suggest that relatively abundant cell types in tissues such as the salivary glands and kidney support transcription of the Py early region (Dawe, 1960). It may be that the viral components missing in these transgenic mice affect the transcriptional specificity as well as the tumor spectrum of the intact virus.

The transgene RNA initiates at two different sites, and one of these corresponds to the major transcriptional start site identified in infected cells and in transformed cell lines (Kamen et al., 1962). Theobservation that in testes the upstream site is used exclusively, while in tumors either site (but not both) is utilized, is suggestive of regulatory com- plexity, although the explanation of this phenomenon is unclear. Nevertheless, the identification of middle T protein in testes and tumors via the kinase assay indicates that both transcripts are functional mRNAs.

Oncogenesis by Middle T Protein The expression of Py middle T protein complexed to a cellular kinase correlates with tumor formation in endothelial cells. The focal nature of the tumors implies that not every endothelial ceil undergoes a proliferative expansion. The short latency and the multiplicity of the tumors, however, suggests that secondary events contributing to tumor formation are not exceedingly rare. Among possible explana- tions for the observed phenotype are that all endotheiial cells express middle T and another genetic or epigenetic event provides a complementing activity in a stochastic fashion, or that a transcriptionally inactive middle T gene is activated in rare endothelial cells, and this expression is sufficient for tumorigenesis. In situ analysis of transgene expression should distinguish between these possi- bilities.

Although most vascuiarized tissues can sustain hemangiomas, the evaluation of Py MT transgenic animals indicates that either liver or lungs are more likely to contain multifocal clusters of the tumors when compared to other tissues. The clustering of tumors in a single tissue suggests a local effect or action. it is possible that endotheiial ceils, which normally proliferate and migrate in a “sprout- like” manner, also proliferate and migrate in this way when tumorigenic to form local interconnected networks of tumor cells. Alternatively, tumorigenesis may induce local growth factor release, which stimulates nearby endotheiial cells to proliferate. The histological analysis of Py MT hemangiomas is consistent with proliferation and local migration (see Figure 3).

Expression of complexed middle T protein in the testes does not lead to tumor formation or detectable abnormalities in Py MT transgenic animals. Although the cell type within the testes supporting Py MT expression has not been determined, it is unlikely that expression is localized to testicular endothelial cells because hemangiomas are rare in testes of Py MT transgenic mice, and because transgene expression is readily detected in testes but not in well-vascularized organs such as liver and kidney. Thus expression of Py middle T antigen in endothelial cells and in testes produced very different results: endotheiiai cell expression leads to proliferation and tumor formation, while testicular expression is inconsequential.

These observations suggest that endothelial cells may be unusually susceptible to the action of the Py MT oncogene. Kornbiuth et al. (1986) have shown that chickens infected with an avian retrovirus containing the Py middle T gene develop endotheliai ceil tumors. Transcription of the middle T gene was presumably regulated by the Rous sarcoma virus LTR because the Py enhancer was not present in the virus. The fact that Py middle T produces hemangiomas in two systems that differ in host species, mode of introduction of the gene, and in the transcriptional control elements regulating expression supports the idea that endothelial cells may be a specific target of the mid- die T oncogene. Other oncogenes appear to selectively perturb specific cell types in transgenic mice: myc is particularly effective in lymphoid and mammary cells (Stewart et al., 1984; Adams et al., 1985; Leder et al., 1986) while fos perturbs cells of the bone lineage (Ruther et al., 1987). Thus tumorigenesis resulting from expression of these oncogenes may require a complementing activity that is unique to the target ceil type.

implications for Vascular Biology Angiogenesis, the formation of new blood vessels, is an important step in tumor progression because it allows microscopic foci of proliferating ceils to expand. Endothelial cells play a crucial role in this process (reviews, Folkman and Klagsbrun, 1987; D’Amore and Thompson, 1987). The regulation of endotheliai cell proliferation is normally very tightly controlled. Once vascularization during embryo- genesis is complete, most endotheliai cells have a dou- bling time on the scale of years. These quiescent cells, however, can be induced to proliferate in a few hours by stimuli produced during the normal process of ovulation, during the traumatic response to wounding, and by tumor ceils during the pathological process of tumor progression. The results presented here demonstrate that the Py middle T oncogene is able to promote pathological vascular angiogenesis in transgenic mice. It is significant that the uterine endothelium of female transgenic mice, although regularly stimulated to proliferate during ovulation and pregnancy, does not give rise to endothelial tumors. This suggests that vascular proliferation is not sufficient to initiate Py middle T induced tumorigenesis. The establishment of this stable strain of mice, which heritably develop endothelial cell tumors, provides a unique format for studying mechanisms of vascular tumorigenesis and the control of angiogenesis.

Experimental Procedures

DNA Constructions The Py DNAs are derivatives of pdPxl3Bla3, which has been modified from the wild-type genome as follows. The Al strain Py genome (Pomerantz et al., 1983) was linearized at the unique BamHl site in the late region for insertion into the vector (pML2, Lusky and Botchan, 1981). The early regulatory sequences are intact, except that a 23 bp deletion and addition of an Xhol linker at bp 3i-60 has removed an LT binding site and renders the genome replication-defective (Pomerantz et al., 1983; Cowie and Kamen. 1984; Py nucleotide numbering is according to Soeda et al., 1980). A Clal linker was inserted at the transcription initiation site (nucleotide 152) and the early coding region was replaced by cDNAs to generate pdPxl3Bla3LTl (Py LT) and

Cell 536

pdPxl3Bla3MT5 (Py MT). The late region carries a large deletion of approximately 500 bp that includes a Hindlll site at position 3918. This deletion precludes the synthesis of the late region structural proteins. PxBP64 was generated by inserting the 1 kb BamHl to Pstl fragment of pdPxl3Bla3 (containing the early region transcription initiation sites) into SP64 in the antisense direction relative to the SP6 promoter.

Microinjection and Establishment of Lines All embryos were F2 hybrids derived from mating B6D2 Fl mice (The Jackson Laboratory). DNAs were linearized within vector sequences with Sall, phenol extracted and EtOH precipitated twice, and passed through a Millipore 0.45 urn small diameter filter. Concentration was determined by ethidium bromide staining of agarose gels containing serial dilutions of samples along with known quantities of DNA. All microinjection procedures and oviduct transfers were done as described by Costantini and Lacy (1982) and Hogan et al. (1986). Briefly, the pronuclei of fertilized one-cell embryos were microinjected with approximately 2 pl (100-200 copies) of DNA, the surviving embryos (80%-90%) were placed into the oviducts of pseudopregnant females and allowed to develop to term (IO%-20% of transferred embryos). The presence of the integrated transgene was assayed by preparing genomic DNA from tail biopsies of 14-day mice (Bautch, 1986). The DNA was digested with Hindlll, subjected to agarose gel electrophoresis, and transferred to filters and hybridized as described (Bautch, 1986). The probe was 32P-pdPX13Bla3 DNA made radioactive by nick translation (Rigby et al., 1977). Positive mice were mated to either C57BUW or DBA/2J mice, and the approximate copy number of the transgene was determined by comparing the intensity of the hybridizing signal in the resulting heterozygotes with dilutions of the original plasmid DNA. These lineages have been maintained in the heterozygous state for one year.

RNA Isolation and Protection Assay Two procedures were used to isolate RNA. All tissues were flash-frozen in liquid N2 and stored at -70°C. Initially RNA was isolated by using a Dounce homogenizer lo disrupt tissue in a 7 M urea/2% SDS ly- sis buffer. The homogenate was extracted with phenol/CHCIS (l:l), CHC13, and pelleted through a 5.7 M CsCl cushion (Noyes et al., 1980). Because this procedure gave variable yields of small tissues. later RNAs were isolated using a modification of the hot phenol method (Maniatis et al.. 1982). Tissues were disrupted using a polytron (Tek- mar) in 4 M guanidine isothiocyanate. heated to 6OpC, diluted in one- third volume 0.1 M NaAc, 10 mM Tris-Cl (pH 7.4), I mM EDTA, and incubated with phenol and phenoUCHCI3 (1 :l) for 10 min at 60°C. The aqueous phase was extracted with phenol/CHC13 and CHC13 at room temperature, and precipitated with EtOH. The pellets were resuspended in 300 mM NaCI. 10 mM T&Cl (pH 7.6), 5 mM EDTA, 0.5% SDS, 250 bglml proteinase K, and incubated at 37°C for 1 hr. After phenol extraction and EtOH precipitation. the pellets were resuspended in 50 mM Tris pH (7.5), 10 mM MgCI,, 5 mM CaC12, 4 @ml DNAase. and incubated at 3PC for 1 hr. The RNAs were digested with 250 wglml proteinase K in the presence of 10 mM EDTA and 0.5% SDS for 15 min at 3PC, phenol extracted, and precipitated with E1OH. The pellets were resuspended in H20 and quantitated by measuring the ODps,,.

The RNA protection assay was performed by hybridizing 10 or 20 pg of total RNA from mouse tissues to an excess of PxBP64 probe, pre- pared using SP6 polymerase, followed by digestion with RNAases A and Tl as described (Zinn et al., 1983; Melton et al., 1984). The RNAase protected RNAs were analyzed by electrophoresis through 8 M urea/5% polyacrylamide thin sequencing gels.

Kinaae Assays Mouse tissues flash-frozen in liquid N2 and stored at -70°C were disrupted with a Dounce homogenizer in RIPA buffer (Lipsich et al., 1983), and the kinase assay was performed on equal amounts of total protein as described (Lipsich et al., 1983), except that protein A sepharose was added to the antigen-antibody complex. Monoclonal antibody 815 (gift from J. Bolen) was used to detect Py MT antigen, and monoclonal antibody 327 (gift from J. Brugge) was used to detect c-src. The immuno- precipitated proteins were electrophoresed through SDS/7.5% polyacrylamide gels. The gels were fixed in 30% MeOH/7% HAc, then incubated in 1 N KOH at 55OC for 1 hr with gentle shaking. The base was neutralized with an equal volume of 1 N HCI, and the gel was rinsed in 30% MeOH before drying.

In Vivo Paaaage of Tumors Tumors were passaged in 6-10 week old B6D2 Fl females. Tumors were carefully excised from the transgenic animal, minced with scis- sors, and rinsed in three changes of sterile saline. The tumor pieces were further minced with sterile blades and sheared by forcing them through a syringe. The final mixture in sterile saline was injected sub- cutaneously into Fl animals anesthesized with Avertin (Hogan et al., 1986).

Acknowledgments

We thank J. Folkman for advice and encouragement and S. Alpert, T. Grodzicker, and W. Herr for helpful discussions and critical reading of the manuscript. We thank J. Brugge and J. Bolen for monoclonal antibodies, and V. L. B. thanks J. Brugge for assistance with the kinase assay, W. Herr for assistance with the RNA protection assay, and J. Skowronski for assistance with tumor passage. We thank S. Minkowitz and I. Seidman for histopathological analysis, B. Lu for technical assistance, D. Biedermann, L. DiLacio, and C. O’Loughlin for animal care, M. Goodwin for typing the manuscript, M. Ockler and J. Roberts for artwork, and D. Greene for photography.

This research was supported by a grant from the Monsanto Com- pany to Cold Spring Harbor Laboratory, and institutional ACS grant award to V. L. B., and a grant from the National Cancer Institute of Canada lo J. A. H. J. A. H. is a Terry Fox Cancer Research Scientist of the National Cancer Institute of Canada. V. L. B. was supported by a fellowship from New York State Health Research Council (#D3-042).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 23, 1987; revised August 25, 1987.

References

Abramczuk. J., Pan, S., Maul, G., and Knowles, B. B. (1984). Tumor induction by simian virus 40 in mice is controlled by long-term persis- tence of the viral genome and the immune response of the host. J. Virol. 49, 540-548.

Adams, J. M., Harris, A. W., Pinkert, C. A., Cocoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R. L. (1985). The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318, 533-538.

Bautch, V. L. (1986). Genetic background affects integration frequency of ecotropic proviral sequences into the mouse germ line. J. Virol. 60, 693-701.

Bishop, J. M. (1985). Viral oncogenes. Cell 42, 2338.

Bishop, J. M. (1987). The molecular genetics of cancer. Science 235, 305-311.

Bolen, J. B., Thiele. C. J., Israel, M. A., Yonemoto, W., Lipsich, L. A., and Brugge, J. S. (1984). Enhancement of cellular src gene product as- sociated tyrosyl kinase activity following polyoma virus infection and transformation. Cell 38, m-777.

Brinster, R. L., Chen, H. Y.. Messing, A., van Dyke, T.. Levine, A. J., and Palmiter, R. D. (1984). Transgenic mice harboring SV-40 T antigen genes develop characteristic brain tumors. Cell 37, 367-379.

Costantini, F., and Lacy, E. (1982). Gene transfer into the mouse germ- line. J. Cell. Physiol. Suppl. 1, 219-226.

Courtneidge, S. A., and Smith, A. E. (1983). Polyoma virus transforming protein associates with the product of the c-src cellular gene. Na- ture 303, 435-439.

Cowie. A., and Kamen, R. (1984). Multiple binding sites for polyomavirus large T antigen within regulatory sequences of polyomavirus DNA. J. Virol. 52, 750-760.

D’Amore. P A., and Thompson, R. W. (1987). Mechanisms of angiogenesis. Ann. Rev. Physiol. 49, 453-464.

Dawe. C. J. (1960). Cell sensitivity and specificity of response to polyoma virus. Natl. Cancer Inst. Monogr. 4, 67-128.

Dawe. C. J., Freund. R., Mandel. G., Ballmer-Hofer, K., Talmage, D. A., and Benjamin, T. L. (1987). Variations in polyoma virus genotype in re- lation to tumor induction in mice. Amer. J. Path. 72i! 243-261.


Eddy, B. E. (1962). Polyomavirus. In The Mouse in Biomedical Re- search, Volume II, H. L. Foster, J. D. Small, and J. G. Fox, ads. (New York, Academic Press), pp. 293-311.

Farber, E., and Cameron, Ft. (1960). The sequential analysis of cancer development. Adv. Cancer Res. 31, 125-226.

Fenton, R. G., and Basilica, C. (1962). Changes in the topography of early region transcription during polyoma virus IyIic infection. Proc. Natl. Acad. Sci. USA 79, 7I42-7I46.

Folkman, J., and Klagsbrun, M. (1967). Angiogenic factors. Science 235, 442-447.

Freund, R., Mandel, G., Carmichael, G. G., Barncastle, J. P., Dawe, C. J., and Benjamin, T L. (1967). Polyomavirus tumor induction in mice: influences of viral coding and non-coding sequenceson tumor profiles. J. Virol. 67, 2232-2239.

Gross, L. (1970). The parotid tumor (polyoma) virus. In Oncogenic Viruses, 2nd edition (Oxford: Pergamon), pp. 651-750.

Hanahan, D. (1965). Heritable formation of pancreatic 5 cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 375, 115-122.

Hanahan, D. (1966). Oncogenesis in transgenic mice. In Oncogenes and Growth Control, P Kahn and T. Graf, eds. (Berlin: Springer-Verlag). pp. 349-363.

Hargis, B. J., and Malkiel, S. (1979). Sarcomas induced by injection of Simian virus 40 into neonatal CFW mice. J. Natl. Cancer Inst. 63, 965-867.

Hogan, B., Costantini, F, and Lacy, E. (eds). (1986). Manipulating the Mouse Embryo: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).

Kamen, R., Jat, P, Treisman, R., and Favaloro, J. (1962). 5’termini of polyoma virus early region transcripts synthesized in viva by wild-type virus and viable deletion mutants. J. Mol. Viol. 759, 169-224.

Klein, G., and Klein, E. (1965). Evolution of tumours and the impact of molecular oncology. Nature 315, 190-195.

Knudson, A. F., Jr. (1966). Geneticsof human cancer. Ann. Rev. Genet. 20, 231-251.

Kornbluth, S., Cross, F. R., Harbison, M., and Hanafusa, H. (1966). Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector. Mol. Cell. Biol. 6, 1545-1551.

Kornbluth, S., Sudol, M., and Hanafusa, H. (1987). Association of the polyomavirus middle-T antigen and c-yes protein. Nature 325, 171- 173.

Lacey, M., Alpert, S., and Hanahan, D. (1966). Bovine papillomavirus genome elicits skin tumours in transgenic mice. Nature 322,609-612.

Land, H., Parada, L. F., and Weinberg, R. A. (1963). Tumorigenic con- version of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304, 596-602.

Leder, A., Pattengale. P K., Kuo, A., Stewart, T. A., and Leder, P (1966). Consequences of widespread deregulation of the C-EC gene in trans- genie mice: multiple neoplasms and normal development. Cell 45, 465-495.

Lipsich, L. A., Lewis, A. J., and Brugge, J. S. (1963). Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J. Virol. 48, 352-360.

Lusky, M., and Botchan, M. (1961). Inhibition of simian virus 40 replication in simian cells by specific pBR322 DNA sequences, Nature 293, 79-61.

Mahon, K. A., Chepelinsky, A. B., Khillan, J. S., Cverbeek, P A.. Pi- atigorsky, J., and Westphal, H. (1967). Oncogenesis of the lens in trans- genie mice. Science 235, 1622-1626.

Maniatis, T., Fritsch, E. F., and Sambrook, J. (1962). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Har- bor Laboratory).

Melton, D. A., Krieg, P A., Rebagliati, M. R., Maniatis, T., Zinn, K., and Green, M. R. (1984). Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacte- riophage SP6 promoter. Nucl. Acids Res. 12, 7035-7056.

Muller, W. J., Mueller, C. R., Mes, A.-M., and Hassell, J. A. (1963). Poly-

omavirus origin for DNA replication comprises multiple genetic elements. J. Virol. 47, 566-599.

Noyes, B. E., Mevarech, M., Stein, R., and Agarwal, K. L. (1979). Detec- tion and partial sequence analysis of gastrin mRNA by using an oligodeoxynucleotide probe. Proc. Natl. Acad. Sci. USA 76, 17701774.

Ornitz, D. M., Palmiter, R. D., Massing, A., Hammer, R. E., Pinkert, C. A., and Brinster, R. L. (1986). Elastase I promoter directs expression of human growth hormone and SV40 T antigen genes to pancreatic aci- nar cells in transgenic mice. Cold Spring Harbor Symp. Quant. Biol. 50, 399-409.

Palmiter, R. D., and Brinster, R. L. (1966). Germ-line transformation of mice. Ann. Rev. Genet. 20, 465-499.

Palmiter, R. D., Chen, H. Y., Messing, A., and Brinster, R. L. (1965). SV40 enhancer and large-T antigen are instrumental in development of choriod tumours in transgenic mice. Nature 376, 457-460.

Pomerantz, B. J., Mueller, C. R., and Hassell, J. A. (1983). Polyomavi- rus large T antigen binds independently to multiple, unique regions on the viral genome. J. Virol. 47, 600-610.

Rassoulzadegan, M., Cowie, A., Carr, A., Glaichenhaus, N., Kamen, R., and Cuzin, F. (1962). Therolesof individual polyomavirus early proteins in oncogenic transformation. Nature 300, 7t3-7t6.

Rigby, P W., Diekmann, M., Rhodes, C., and Berg, P. (1977). Labeling deoxyribonucleic acid to high specific activity in vitro by nick-translation with DNA polymerase I. J. Mol. Biol. 173, 237-251.

Ruley, H. E. (1963). Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 304, 602-607.

Ruther, U., Garber, C., Komitowski, D., Muller, R., and Wagner, E. F. (1987). Deregulated c-fos expression interferes with normal bone development in transgenic mice. Nature 325, 412-416.

Sinn, E., Muller, W., Pattengale, P, Tepler, I., Wallace, R., and Leder, P (1967). Coexpression of MMTV/v-Ha-ras and MMTVlc-myc genes in transgenic mice: synergistic action of oncogenes in vivo. Cell 49, 465-475.

Small, J. A., Khoury, G., Jay, G., Howley, P M., and Scangos. G. A. (1966). Early regions of JC virus and BK virus induce distinct and tissue-specific tumors in transgenic mice. Proc. Natl. Acad. Sci. USA 83, 6288-8292.

Soeda, E., Arrand, J. R., Smolar, N., Walsh, J. E., and Griffin, B. E. (1980). Coding potential and regulatory signals of the polyoma virus genome. Nature 283, 445-453.

Stewart, T. A., Pattengale, P K., and Leder, P (1984). Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTVlmyc fusion genes. Cell 38, 627-637.

Tooze, J. (1981). Molecular Biology of Tumor Viruses. Part 2, DNA Tu- mor Viruses (Cold Spring Harbor, New York: Cold Spring Harbor Labo- ratory).

Treisman, R.. Cowie, A., Favaloro. J., Jat, P., and Kamen. R. (1981a). The structure of the spliced mRNAs encoding polyoma virus early region proteins. J. Mol. Appl. Genet. 7, 63-92.

Treisman, R., Novak, U., Favaloro. J., and Kamen, R. (1981b). Transfor- mation of rat cells by an altered polyoma virus genome expressing only the middle-T protein. Nature 292, 595-600.

Weinberg, R. A. (1985). The action of oncogenes in the cytoplasm and nucleus. Science 230, 770-776.

Zinn, K., DiMaio. D.. and Maniatis, T. (1983). Identification of two distinct regulatory regions adjacent to the human p-interferon gene. Cell 34, 865-879.

Note Added in Proof

The susceptibility of vascular endothelial cells lo the action of Py MT is supported by the work of Williams, Courtneidge, and Wagner (Cell, submitted), who have shown that Py MT antigen can cause embryonic lethalities and endothelial tumors in chimeric mice obtained from retrovirally infected embryonic stem cells.

endothelial cell tumors develop in transgenic mice carrying polyoma virus middle t oncogene

Documents