VIROLOGY
99,
135- 144
Distribution R. KOSHY,” *Laboratory
(1979)
of Feline Leukemia Virus DNA Sequences Normal and Leukemic Domestic Cats F. WONG-STAAL,*
R. C. GALLO,“,’
W. HARDY,t
in Tissues
AND M. ESSEXS
of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20205; TMemorial Kettering Cancer Center, New York, New York 10021; and SDepartment of Microbiology, Harvard University School of Public Health, Boston, Massachusetts 02115 Accepted
July
of
Sloan-
16, 1979
Seroepidemiological studies indicate that leukemia and lymphoma in cats are horizontally transmitted and that feline leukemia virus (FeLV) is the agent responsible for the disease. Yet, in a significant proportion of leukemic cats, FeLV has not been identified, despite the presence of the feline oncornavirus-associated cell membrane antigen (FOCMA) and in some instances, epidemiological evidence indicating exposure to FeLV. Prompted by these so called virus negative cases of cat leukemia, we surveyed a total of 176 tissues from 50 cats for the presence of FeLV proviral DNA sequences. The animals studied included viremic and nonviremic cats that were either healthy or leukemic. We found: (1) DNA from virus-positive tissues from both healthy and leukemic cats hybridized 60-100% of lZ51-FeLV (strain Rickard) RNA. (These values are normalized to hybrid yield obtained with DNA of cat cells infected with FeLV in culture, which is 55%.) (2) A few virus-negative tissues also hybridized 60-100% of FeLV RNA. These tissues were derived from lymphomabearing cats as well as healthy cats. (3) The majority of virus-negative tissues hybridized 30-60% of FeLV RNA, and in this group, tissues from leukemic cats did not show significantly higher hybridization. (4) The results did not define a particular tissue target site since comparable hybridization results were obtained with many different tissues. (5) A few virus-negative tissues from healthy cats hybridized only 15-30% FeLV RNA. This is considerably lower than the level found with the majority of virus-negative tissues (normal or leukemic) and may reflect the true level of endogenous FeLV related sequences in cats. If so, the higher hybridization levels (30-60% of FeLV RNA) observed with most virusnegative tissues indicates more widespread infection in cats than previously believed, and suggests integration of only partial provirus and/or infection of only a fraction of the cells in the tissue. If exogenous FeLV is involved in the leukemogenesis of “virus-negative” cats, the apparent lack of detectable proviral sequences in tissues of many of these cats over and above those of healthy cats could be interpreted in different ways, e.g., partial loss of provirus, integration of a small fragment, or presence of proviral sequences in a limited population of cells. These “virus-negative” cats may serve as a model (S. M. Cotter and M. Essex, 1977, Amer. J. Pathol. 87,265-268) for other animals, including man, where leukemia may sometimes be associated with certain type-C related subviral markers, but detection of provirus is unusual. INTRODUCTION
are easily infected with FeLV when in the animals, and Many naturally occurring spontaneous company of virus-excreting when infected, they have a much higher risk leukemias and lymphomas in the domestic of developing leukemia and lymphomas cat (Felis catus) are caused by a type-C RNA tumor virus, feline leukemia virus (Hardy et cd., 1973b; Essex et aE., 1975, 1977a). The majority of infected cats with (FeLV), which is transmitted by horizontal leukemia and lymphomas have readily deinfection (Essex, 1975; Hardy et al., 1973; tectable free FeLV or viral antigens in their W. F. Jarrett et al., 1973). Uninfected cats tissues (Essex et al., 1975; Hardy et al.. 1973). There have be& some studies evalu: ating the presence of detectable FeLV re1 To whom reprint requests should be addressed. 135
0042-6822/79/150135-10$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
136
KOSHY
lated sequences in feline DNA. Gillespie, et al. (1973) demonstrated the presence of FeLV related sequences in the spleens of a few specific-pathogen-free normal cats. Quintrell et al. (1974) obtained about 30% hybridization of FeLV RNA to DNA from the livers of three normal cats which were negative for FeLV antigens. Okabe et al. (1976) showed FeLV related DNA in cat tissues including the livers of two specificpathogen-free cats. Benveniste et al. (1975) have reported that sequences partially related to the RNA of FeLV are present endogenously in all cats including specificpathogen-free animals. Levin et al. (1976) reported the presence of FeLV related sequences in six virus-negative tissues studied. Approximatley 30% of cats with spontaneous leukemia have neither free virus nor serologically assayable viral components (Hardy et al., 1969; Hardy, 1971; Essex, 1975). However, two lines of evidence suggest that FeLV might nonetheless be involved in the etiology of these virus-negative tumors: (a) The incidence of virus-negative cases of leukemia in cats that live in leukemia cluster households with virus-positive excretor cats is significantly higher than of those in carrier-free households (Essex et al., 1979); (b) feline oncornavirus associated cell-membrane antigen (FOCMA) which is a transformation-specific antigen known to be encoded by the related feline sarcoma viruses (FeSV) (Sliski et al., 1977; Stephenson et al., 1977; Sherr et al., 1978) is expressed in virus-negative leukemic cells (Hardy et al., 1977; Essex et al., 197713, 1979). It is this group of cats that we are particularly interested in because of possible similarities to other virus-negative species, especially man, where type-C virus related subviral molecules have sometimes been observed in association with leukemia (reviewed by Gallo and Meyskens, 1978). In naturally infected chickens (Neiman et al., 1975), cattle (Miller et al., 1976), and some primates (Scolnick et al., 1974; Gallo et al., 1978), it has been shown that tumor tissues contained retroviral sequences that normal tissues did not, suggesting transmission of acquired virus among these animals. Using DNA complementary (cDNA)
ET AL.
to subgroup B of FeLV (FeLV-B), Levin et al. (1976) observed extra copies of FeLV DNA in virus-positive tumor tissue that were not present in normal tissues, while no difference between normal and tumor tissue was detected in virus-negative cats. However, comparison was not made between normal and tumor tissues from the same cat. In this initial study we were interested in examining DNA from many tissues from each of a large number of cats for FeLV related sequences. Since we are studying field animals where the nature of the infecting agent may vary from one animal to the next, the choice of a particular subgroup of FeLV for probes appears arbitrary. However, of the three subgroups, FeLV-A is the most virulent strain, and is the most commonly occurring in nature. FeLV-A is always present in virus-positive cats with leukemia or lymphoma and can occur alone in a significant proportion of the cases, while FeLV-B or FeLV-C is always accompanied by FeLV-A, (Hardy et al., 1976; 0. Jarrett et al., 1978; Sarma and Log, 1973). Consequently, FeLV-A was used as probe. The results show that virus-positive cats almost invariably have greater amounts of hybridizable sequences than virusnegative cats, but with no apparent preferred target tissue for virus integration. However, there were no significant differences between normal and leukemic tissues in virus-positive or in virus-negative cats. Finally, DNA from a few virus-negative tissues from a few cats contained considerably lower levels of FeLV related sequences than all other samples. MATERIALS
AND
METHODS
Feline tissues. Tissues were obtained at autopsy from pet cats which had developed leukemidlymphoma, or from normal cats from the Animal Medical Center, New York; and the Angel1 Memorial Animal Hospital, Boston. Several specific-pathogen-free kittens were purchased from Liberty Laboratories, Liberty Corner, New Jersey. A total number of 176 tissues from 50 different cats were studied. The tissues were carefully examined by
FeLV
RELATED
SEQUENCES
light microscopy and in all cases there was no extensive infiltration of tumor cells in non-tumor tissues, and the tissues were not necrotic. Of the 50 cats, 14 were FeLV negative and healthy, 21 were FeLV negative and leukemic, 3 were FeLV positive and healthy, and 12 were FeLV positive and leukemic. The cats were classified as “virus positive” or “virus negative” by means of the immunofluorescence test (Hardy et al., 1973a,b) which detects expression of FeLV in the peripheral blood. Extraction of DNA. Fresh or frozen cat tissues were minced and homogenized in a Sorvall Omnimix homogenizer with 3 vol of 10 mM EDTA and 50 mM Tris, pH 9.0. The homogenates were treated with 1% sodium dodecyl sulfate (SDS, GallardSchlessinger) for 20 min at 60”. They were then extracted first with phenol:chloroform: isoamyl alcohol (1:1:0.2) and then with chloroform alone, precipitated with 2 vol of ethanol at -2O”, redissolved in 50 n-&f Tris (pH 9.0) and 0.5% SDS, and digested with Pronase (300 pug/ml). The DNA was reextracted as before and precipitated again with ethanol. DNA precipitates were dissolved in distilled water and sheared by sonication to an average size of 400 nucleotides. The sheared DNA was treated with 0.3 N NaOH for 18 hr at 37”, and then neutralized with sodium acetate (pH 4.5). Then, the DNA was precipitated with ethanol, collected by centrifugation in a RCB-B Sorvall centrifuge, and dissolved in water. Viruses. Subgroup A feline leukemia virus (FeLV), strain Rickard, was grown in feline embryo fibroblasts and concentrated from culture medium by centrifugation followed by equilibrium sedimentation in sucrose gradients. PurQication of viral RNA. FeLV was solubilized with 1% SDS in the presence of Pronase (300 gg/ml). RNA was extracted repeatedly with redistilled phenol (saturated with 50 m&f Tris pH 9.0) until the ratio of optical density at 260 rnp to that at 280 rnp was greater than 1.9. The RNA was then fractionated by ultracentrifugation on lo-30% glycerol gradients in a SW 40.1 rotor at 40,000 rpm for 3 hr at 4”. The ‘70 S RNA from the gradient was
IN DNA
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precipitated with ethanol at -20” overnight and collected by centrifugation at 12,300 gin a Sorvall RCB-B centrifuge. Radioiodination of viral RNA was kindly performed by Dr. W. Prensky, Memorial Sloan-Kettering Cancer Center, New York. Following iodination, the RNA samples were precipitated with cetyltrimethylammonium bromide (Reitz et al., 1972). Precipitates were dissolved in 1 M sodium chloride and reprecipitated with ethanol at -20” overnight. The precipitates were collected by centrifugation and dissolved in 10 m.J4 Tris-HCl pH 7.0. Hybridization of nucleic acids. lz51-FeLV (Rickard) RNA, lo-20 pg (i.e., -1500 cpm), was added to 100 pg of cat DNA in a final volume of 12 ~1 containing 0.4 M sodium phosphate, pH 6.8. The mixtures were sealed in capillary pipets, boiled for 5 min, and incubated to a C,t of lo4 (uncorrected for [Na+]>. Hybrids were assayed as previously described (WongStaal et aZ., 1976) by RNase A digestion (50 pug/ml, 0.6 M NaCl, 1 hr, 37”). Percentage hybridization was calculated, and in each case, normalized to the value obtained by hybridizing lz51-FeLV RNA to DNA of tissue cultured cat cells infected with FeLV (55%). Repeat assays for several samples selected randomly yielded comparable values. All samples were received coded, and the diagnosis and viral status of the animals were known only after the hybridization values were obtained. Hybridization controls. Several samples from each range of hybridization were checked for ability to hybridize with lz51ribosomal RNA and lz51-RD114 RNA. Ribosomal RNA was a mixture of 28 and 18 S purified from HeLa cells and was a gift from Dr. W. Prensky. RD114 RNA was gradient purified 70 S RNA from virus grown in RD cells. The lz51-FeLV RNA used in the hybridizations were checked by hybridization to DNA obtained from livers of leopard cat (Felis bengalensis) and baboon (Papio cynocephalus). The leopard cat is a distant relative of the domestic cat, having cellular sequences in common with it, but not FeLV related sequences (Benveniste et al., 1975). Baboon DNA has endogenous viral sequences related to RD114 virus, an endog-
138
KOSHY
enous virus in the domestic cat (Benveniste et al., 1975). Neither leopard cat DNA nor baboon DNA hybridized more than 10% of lz51-FeLV RNA, thus indicating that the viral probe was reasonably free of contaminating feline cellular or RD114 viral sequences. Kinetics of hybridization. Hybridization reactions were set up as described above. Each DNA sample was incubated to different lengths of time (0 to 100 hr) to give C,t values of 0 to 104, respectively. The hybrids were analyzed as described previously (Wong-Staal et al., 1976) and the normalized values plotted. Thermal melting of hybrids. Hybridizations were done as described above. The hybrids were melted for 2 min each at the indicated temperature and assayed as before. A graph was plotted of the temperature of melting versus the percentage of hybrids remaining. The melting temperature (t,) was the temperature at which 50% of the RNA remained in hybrid form. RESULTS
Hybridization of Y-FeLV to Cat DNA
(Rickard)
ET AL.
between healthy and leukemic cats. Six tissues from a few nonviremic cats hybridized in the LOW range. These results and possible interpretations of them are detailed in the following. Virus-Positive Cats: Lack Target Tissue for Virus
of Preferred Replication
Hybridization values obtained with various tissues of individual viremic cats are presented in Fig, 1. DNA from almost all of the tissues, whether derived from healthy or leukemic cats, hybridized in the HIGH range. Surprisingly, neither the tumor tissues nor any other tissue hybridized FeLV RNA preferentially. These results indicate that there are no preferred target tissues, including the tumor tissues, for virus replication in the viremit cats. Although viral sequences seemed ubiquitous in viremic cats, one tissue appeared to be restrictive for virus replication. In four out of six cats in which DNA from muscle was examined, this DNA hybridized significantly less FeLV RNA (Fig. 1).
RNA
A total of 176 tissues from 50 cats were studied for FeLV related sequences in their DNA. Hybridization of lz51-FeLV RNA to DNA from leopard cat and from baboon was less than lo%, showing respectively that the FeLV RNA used as a probe was relatively free of contaminating feline cellular sequences as well as RD114 viral sequences. Four groups of cats were studied: nonviremic-healthy, nonviremic-leukemic, virem&healthy, and viremic-leukemic. For convenience of comparison between the groups, hybridization values are designated HIGH @O-100%), INTERMEDIATE (30SO%), and LOW (15-30%). DNA from most tissues of viremic cats hybridized in the HIGH range. Some tissues of nonviremic cats, both healthy and leukemic, also hybridized in the HIGH range, with no ap preciable difference in the levels between healthy and leukemic cats. The majority of tissues from nonviremic cats, however, hybridized in the INTERMEDIATE range, again with no significant differences
Virus-Negative
Cats
Leukemic versus healthy animals. DNA from a few tissues of virus-negative eats hybridized in the HIGH range. This was true for tissues from both healthy and leukemic cats (Figs. 2 and 3). DNA from the majority of tissues from virus negative cats, healthy or leukemic, hybridized in the INTERMEDIATE range. Thus, no increased amounts of proviral sequences were seen in tumor tissues of virus-negative cats compared to normal tissues of healthy virusnegative cats. On the basis of the results of this survey of multiple tissues from a large number of cats, it is unlikely that a nontarget (nontumor) tissue serves as a reservoir for virus integration and replication. Low hybridization values. A surprising result was that DNA from five tissues of four healthy cats and one tissue from a leukemic eat hybridized considerably lower amounts of FeLV RNA (l&30%) than DNA from all other cats (Figs. 2 and 3). These reactions were characterized by ki-
FeLV RELATED
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IN DNA OF CAT TISSUES
139
FIG. 1. Hybridization of lZ51-FeLV-A (Rickard) RNA to DNA from virus-positive healthy and viruspositive leukemic cats. Various tissues from each cat (number N, or L,) were studied. Reactions were done in liquid at DNA excess, 0.4 M sodium phosphate and 60” to a C$ of lo4 (uncorrected for salt concentration). Values are normalized to that obtained by hybridizing FeLV-A RNA to DNA from feline cells infected with FeLV-A, and producing virus.2 DNA from control tissues (viz., from human, baboon, and leopard cat) hybridized FeLV-A RNA less than 1O%.2 Hybridization values are arbitrarily divided into three ranges, 10-30, 30-60, and 60-loo%.* N, normal cats; L, leukemic cat; BM, bone marrow; BR, brain; K, kidney; LI, liver; LN, lymph node; LU, lung; M, muscle; OM, omentum; S, spleen; SG, salivary gland; TF, thoracic fluid; TH, thymus; TO, tongue; TU, tumor.
netics and t, experiments as well (see following section). As an additional control, the quality of the DNA samples was checked by hybridization to labeled ribosomal RNA and RD114 viral RNA. The hybrid yields obtained for ribosomal RNA and for RD114 viral RNA were comparable with DNA samples from the LOW, INTERMEDIATE, or HIGH groups. It is, therefore, unlikely that the LOW values obtained for these samples with FeLV RNA were due to nonspecific inhibitors of hybridization such as contaminating nucleases in the DNA or due to degradation of the DNA. Characterization of the Hybrids by Kinetics and Therm& Stability Measurements
Division of the DNA samples into HIGH, INTERMEDIATE, and LOW ranges was substantiated by hybridization kinetics and thermal stability measurements of the hybrids formed. Figure 4 presents the results 2 Also applies to Figs. 2 and 3.
of kinetic experiments performed with DNA samples from the three different ranges, with DNA from feline cells (FEA) infected with FeLV-A as a positive control. The. results of these experiments corresponded closely with those of the first series of hybridization experiments. Samples from the different groups hybridized again as HIGH, INTERMEDIATE, or LOW. The results indicate that a complete provirus identical to FeLV-A is not present in DNA of all cat tissues as an endogenous virogene. A provirus at even one copy per cell hybridizes 80% of the viral RNA by a C$ of 104, with a C&2 of 5 x lo3 (Wong-Staal et al., 1976), which is more than the extents of hybridization obtained with DNA in either the LOW or INTERMEDIATE groups. Our data do not rule out the possibility of a few cells in a population containing complete FeLV-A provirus, or all cells containing a complete virus partially related to FeLV-A. Within each group, DNA from tumor or nontumor tissues hybridized with similar
140
KOSHY ET AL.
I N1
I N2
I, N3
I b
%
, N6
, NT
, NE
/ N9
Ii, NlD
, Nil
NV
N11
, NJ4
FIG. 2. Hybridization of lz51-FeLV-A (Rickard) RNA to DNA from virus-negative healthy cats. Reactions were carried out as described under Materials and Methods. BM, bone marrow; BR, brain; H, heart; K, kidney; LI, liver; LN, lymph node; LU, lung; M, muscle; S, spleen; SG, salivary gland; TH, thymus; TO, tongue.
kinetics, suggesting that there is no amplification of viral sequences in tumor tissues. Thermal stabilities of the hybrids formed between 1251-FeLV-A RNA and several DNA samples from each range of hybridization were also measured (Fig. 5). The t, values obtained correlated with the extents of hybridization. Thus, samples which hybridized HIGH had the same t, as
DNA from cat cells infected with FeLV-A (ht, = O”). The INTERMEDIATE samples displayed a lower t, (A&, = 29, and the LOW samples had the lowest t, (At, = 5”). DISCUSSION
A great deal of evidence indicates that FeLV is involved in the cause of naturally
FIG. 3. Hybridization of ‘7-FeLV-A (Rickard) RNA to DNA from virus-negative leukemic cats. The reactions were done as described under Materials and Methods. See Fig. 2 for symbol legends.
FeLV RELATED
SEQUENCES
occurring feline leukemia among virus-positive cats. However, approximately 30% of all leukemic cats are virus negative, and the role of FeLV, if any, in leukemia of these cats is not understood. Two separate lines of evidence suggest that leukemia in virus-negative cats may also be induced by FeLV: (a) Leukemic cells of these cats express FOCMA, a tumor-specific antigen induced by FeLV and FeSV (Hardy et al., 19’7’7; Essex et al., 1979; Sliski et al., 19’77). However, since radiation or chemically induced feline leukemia models have not yet been developed and since FeLV has not yet been directly shown to code for FOCMA! this observation does not exclude the possibility that FOCMA is a cell antigen induced by FeLV, and might also be activated by nonviral leukemogens. (b) The incidence of leukemia in virus-negative cats known to be exposed to FeLV is as high as that among exposed cats which become viremic. This increase is greater than would be expected due to chance alone (Essex et al., 1978). Several studies have been conducted to evaluate the presence of FeLV provirus sequences in healthy and leukemic cats (Gillespie et al., 1973; Quintrell et aE., 1974; Okabe et al., 1976; Benveniste et al., 1975; Levin et al., 1976). However, these only
FIG. 4. Kinetics of hybridization of lZ51-FeLV-A RNA to DNA from various cat tissues. Reactions were done as described under Materials and Methods. x - - - x Feline embryo cells infected with FeLV-A. l AOvm Tissues from INTERMEDIATE hybridization group. OV Tissues from INTERMEDIATE hybridization group. DA Tissues from LOW hybridization group.
141
IN DNA OF CAT TISSUES
80 P ?._ E d ; g-? 0
60
40
20
0
60
70
80 Temperature
90
100
IQ
FIG. 5. Thermal melting profiles of hybrids of lZ51-FeLV-A RNA and cat cellular DNA. Hybrids were assayed as described under Materials and Methods. Profiles shown are representative of several samples with “HIGH,” “INTERMEDIATE,” and “LOW” hybridization, respectively. + Feline embryo cells infected with FeLV-A; t, = 82”. n HIGH hybridization; t, = 82”. A INTERMEDIATE hybridization; t, = 80”. 0 LOW hybridization. t, = 77”.
surveyed a small number of samples and usually included only tumor tissues from the leukemic cats. The interpretations were further complicated by the difficulty in distinguishing between endogenous and acquired FeLV related sequences in DNA of cats. In order to understand better the roles of FeLV in leukemogenesis in both viremic and nonviremic cats, and to compare feline leukemia with spontaneously occurring, viral-induced leukemias of other species, we examined DNA from multiple tissues from a large number of cats. These included virus positive-healthy and virus positive-leukemic, virus negative-healthy and virus negative-leukemic cats. The most commonly isolated viruses from cats with lymphomas belong either to subgroup A (FeLV-A) or are mixtures of viruses from subgroup A and subgroup B (FeLV-AB). Subgroup C (FeLV C) is rare and occurs only in mixtures with FeLV A (FeLV-AC) or with FeLV-AB (FeLV-ABC) (Hardy et al., 1976; W. Jarrett et al., 1973; Sarma and Log, 1973). Consequently, RNA from the Rickard strain of FeLV, a virulent strain (Rickard et al., 1969) comprising predominantly subgroup A, was used as the probe.
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KOSHY
The results yielded several interesting observations: (1) The presence of proviral DNA sequences is widespread among the tissues of virus-positive cats, healthy or leukemic, indicating a lack of preferred target tissue(s) for the virus. Among leukemic cats, provirus was detected in almost every tissue in addition to the tumor tissue. This result cannot be due to infiltration of leukemic cells into non-tumor tissues since (a) no extensive infiltration was observed by microscopic examination of the tissues, (b) similar rates of hybridization were obtained with tumor or non-tumor tissues, and (c) in some cases, the DNA from tumor tissues hybridized even less probe than DNA from non-tumor tissues. Similar results have been reported in a case of naturally occurring gibbon ape leukemia, in which proviral sequences of gibbon ape leukemia virus (GaLV) were detected to the same extent in all tissues of the viremic animal except muscle and brain (Gallo et al., 1978). The extent of virus spread may correlate with the level of virus production since both cats and gibbons are high virus excretors. (2) In four out of six viremic animals in which the muscle was examined, DNA from this tissue hybridized considerably lower amounts of FeLV RNA, suggesting that this tissue may be restrictive for virus replication. In the few instances where the muscle contained levels of proviral sequences comparable to those of other tissues, infection may have occurred congenitally. Again the restriction of virus replication in muscle has been observed in viremic gibbons infected postnatally (Gallo et al., 1978; Kawakami et al., 1978). (3) While the majority of virus-negative cat tissues hybridized in the INTERMEDIATE range, DNA from tissues of some “virus-negative” cats (both healthy and leukemic) hybridized the same amount of viral RNA as did virus-positive cats (i.e., 60-100%) (Fig. 2). This extensive hybridization clearly shows that at least in a fraction of the so-called virus-negative cats FeLV infection did take place, even though serological tests failed to indicate this. (4) DNA from some tissues of a small
ET AL.
number of virus-negative cats hybridized unusually LOW amounts of FeLV RNA (15-30%) (Figs. 2 and 3). These results were reproducible and could not be due to degradation of the DNA samples since these DNA samples hybridized ribosomal RNA and RD114 RNA as efficiently as DNA from tissues in the HIGH and INTERMEDIATE range. There are two possible interpretations of these results, which are not mutually exclusive. First, since cats are an outbred species, there may be a wide variation of copy number and divergent genetic loci of the endogenous FeLVrelated virogenes from animal to animal. Second, the LOW values may be the real endogenous FeLV related sequences and the INTERMEDIATE DNA samples contain endogenous as well as novel viral sequences acquired by infection. Results of hybrid analysis by t, determination tend to support the second possibility. Since the LOW level hybrids have the lowest t, values (At, = 5”) the data indicate a divergence of sequences as would be expected for endogenous FeLV related sequences. The INTERMEDIATE level hybrids exhibit t, values intermediate between the LOW and HIGH hybridization samples, consistent with presence of partly endogenous and partly recently acquired sequences. If this hypothesis is correct, then there must be much more widespread infection of cats by FeLV than previously believed since DNA from the majority of virus-negative cats hybridized more viral RNA (INTERMEDIATE range). The lack of complete hybridization in these virus-negative cats could mean that either only part of the provirus is present in the host DNA (due to integration of partial provirus or due to loss of some sequences after integration), that proviral sequences are integrated into the DNA of only a small number of cells, or both. Until recently there was little evidence for incomplete proviral sequences in cells transformed by type-C retroviruses. Svoboda et al. (1977) detected incomplete viral genomes in a mouse nonproducer cell line which had been transformed by Rous sarcoma virus (RSV): Hybridization of the cellular DNA with RSV 35 S RNA yielded a lower plateau
FeLV
RELATED
SEQUENCES
value than was obtained with DNA from a virogenic line. Recently, by means of restriction endonucleases and “blot” hybridization experiments, Wong-Staal et al. (1979) provided direct evidence for partial provirus in DNA from the liver and kidney of a gibbon ape which had been exposed to GaLV. There may be subtle differences between the leukemic and normal virus-negative eats, which cannot be discerned by means of the hybridization techniques used. These differences may become more apparent upon analysis with restriction endonucleases and hybridization of viral probes to specific DNA fragments. Such experiments are now in progress. ACKNOWLEDGMENTS This work was partly supported by NC1 Grants CA13885, CA-16599, CA-18488 and by American Cancer Society Grant DT-32. R.K. is a Fellow of the Fogarty International Center, NIH. W.H. is a scholar of the Leukemia Society of America. REFERENCES BENVENISTE, R. E., SHERR, C. J., ~~~TODARO, G. J. (1975). Evolution of type-C viral genes: Origin of feline leukemia virus. Science 190, 886-888. COWER, S. M., and ESSEX, M. (1977). Animal model: Feline acute lymphoblastic leukemia and aplastic anemia. Amer. J. Pathol. 87, 265-268. ESSEX, M. (1975). Horizontally and vertically transmitted oncornaviruses of cats. Advan. Cancer Res. 21, 175-246. ESSEX, M., COTTER, S. M., HARDY, W. D., HESS, P., JARRETT, W., JARRETT, O., MACKEY, L., LAIRD, H., PERRYMAN, L., OLSEN, R. G., and YOHN, D. S. (1975). Feline oncornavirus-associated cell membrane antigen. IV. Antibody titers in cats with naturally occurring leukemia, lymphoma, and other diseases. J. Nat. Cancer Inst. 55, 463-467. ESSEX, M., CORER, S. M., SLISKI, A. H., HARDY, W. D., STEPHENSON, J. R., AARONSON, S. A., and JARRETT, 0. (1977a). Horizontal transmission of feline leukemia virus under natural conditionsIn a feline leukemia cluster household. Znt. J. Cancer 19, 90-96. ESSEX, M., GRANT, C. K., COTTER, S. M., SLISKI, A. H., and HARDY, W. D. (1979). Leukemia specific antigens: FOCMA and immunosurveillantie. In “Modern Trends in Human Leukemia” (R. Neth, R. C. Gallo, P. H. Hofschneider, and K. Mannweiler, eds.), Pt. III. Springer-Vex-lag, New York, pp. 453-467. ESSEX, M., SLISKI, A. H., HARDY, W. D., DENOR-
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ONHA, F., and GOITER, S. M. (1977b). Feline oncornavirus associated cell membrane antigen: A tumor specific cell surface marker. In “Advances in Comparative Leukemia Research” (P. Bentvelzen et al., eds.), pp. 337-340. Elsevier/North-Holland, Amsterdam. GALLO, R. C., GALLAGHER, R. E., WONG-STAAL, F., AOKI, T., MARKHAM, P. D., SCHETTERS, H., RUSCETTI, F., VALERIO, M., WALLING, M. J., O’KEEFE, R. T., SAXINGER, W. C., GUY SMITH, R., GILLESPIE, D. H., and REITZ, M. S., JR. (1978). Isolation and tissue distribution of type-C virus and viral components from a gibbon ape (Hylobates la?) with lymphocytic leukemia. Virology 84, 359-373. GALLO, R. C. and MEYSKENS, F. L., JR. (1978). Evidence for the presence of RNA tumor virus components in human leukemia. Progr. Exp. Tumor Res. 22, 216-249. GILLESPIE, D., GILLESPIE, S., GALLO, R. C., EAST, J. L., and DMOCHOWSKI, L. (1973). Genetic origin of RD114 and other RNA tumor viruses assayed by molecular hybridization. Nature New Biol. 244, 51-54. HARDY, W. D., JR. (1971). Immunodiffusion studies of feline leukemia and sarcoma. J. Amer. Vet. Med. Ass. 158, 1060-1069. HARDY, W. D., JR., GEERING, G., OLD, L. J., DEHARVEN, E., BRODEY, R. S., and MCDONOUGH, S. (1969). Feline leukemia virus: Occurrence of viral antigen in the tissues of cats with lymphosarcoma and other diseases. Science 166,10191021. HARDY, W. D., JR., HESS, G. W., MACEWEN, E. G., MCCLELLAND, A. J., ZUCKERMAN, E. E., ESSEX, W., COTTER, S. M., and JARRET, 0. (1976). Biology of feline leukemia virus in the natural environment. Cancer RQS. 36, 582-588. HARDY, W. D., JR., HIRSHAUT, Y., and HESS, P. (1973a). In “Unifying Concepts of Leukemia” (R. M. Dutcher and L. Chieco Bianchi, eds.), pp. 778-799. Karger, Basel. HARDY, W. D., JR., OLD, L. J., HESS, P. N., ESSEX, M., and COTTER, S. (197313). Horizontal transmission of feline leukemia virus. Nature NQW Biol. 244, 266-269. HARDY, W. D., JR., ZUCKERMAN, E. E., MACEWEN, G. E., HAYES, A. A., and ESSEX, M. (1977). A feline leukemia virus and sarcoma specific virusinduced tumor-specific antigen. Nature (London) 270, 249-251. JARRETT, W. F., JARRETT, O., MACKEY, L., LAIRD, W., HARDY, W. D., and ESSEX, M. (1973). Horizontal transmission of leukemia virus and leukemia in the cat. J. Nat. Cancer Inst. 51, 833-841. JARRETT, O., HARDY, W. D., JR., GOLDER, M. C., and HAY, D. (1978). The frequency of occurrence of feline leukemia virus subgroups in cats. Znt. J. Cancer 21, 334-337.
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JARRETT, O., LAIRD, H. M., and HAY, D. (1973). Determinants of the host range of feline leukemia viruses. J. Gen. Viral. 20, 169-175. LEVIN, R., RUSCETTI, S. D., PARKS, W. P., and SCOLNICK, E. M. (1976). Expression of feline type-C virus in normal and tumor tissues of the domestic cat. Int. J. Cancer 18, 661-671. MILLER, J. M., MILLER, L. D., OLSON, C., and GILLETTE, K. G. (1969). Virus-like particles in phytohemagglutinin stimulated lymphocyte cultures with reference to bovine lymphosarcoma. J. Nut. Cancer Inst. 43, 1297-1305. NEIMAN, P. E., PURCHASE, H. G., and OKAZAKI, W. (1975). Chicken leukosis virus genome sequences in DNA of normal chick cells and virus induced bursal lymphomas. Cell 4, 311-319. OKABE, H., TWIDDY, E., GILDEN, R. V., HATANAKA, M., HOOVER, E. A., and OLSEN, R. G. (1976). FeLV-related sequences in DNA from a FeLV-free cat colony. Virology 69, 798-801. QUINTRELL, N., VARMUS, H. E., BISHOP, J. M., NICHOLSON, M. O., and MCALLISTER, R. M. (1974). Homologies among the nucleotide sequences of the genomes of C-type viruses. Virology 58, 569-575. REITZ, M. S., ABRELL, J. W., TRAINOR, C. D., and GALLO, R. C. (1972). Precipitation of nucleic acids with cetyltrimethylammonium bromide: A method for preparing viral and cellular DNA polymerase products for cesium sulfate density gradient analysis. Biochem.
Biophys.
Res. Commun.
49, 30-38.
RICKARD, C. G., POST, J. F., DENORONHA, F., and BARR, L. M. (1969). A transmissible virus-induced lymphatic leukemia of the cat. J. Nut. Cancer Inst. 42, 987-996. SARMA, P. S., and LOG, T. (1973). Subgroup classification of feline leukemia and sarcoma viruses by viral interference and neutralization tests. Virology 54, 160-169. SCOLNICK, E. M., PARKS, W. P., KAWAKAMI, T.,
KOHNE, D., OKABE, H., GILDEN, R. V., and HATANAKA, M. (1974). Primate and murine type-C viral nucleic acid association kinetics: Analysis of model systems and natural tissues. 2. Viral. 13, 363-369. SHERR, C. J., SEN, A., TODARO, G. J., SLISKI, A., and ESSEX, M. (1978). Pseudotypes offeline sarcoma virus contain an 85,000.dalton protein with feline oncornavirus-associated cell membrane antigen (FOCMA) activity. Proc. Nut. Acud. Sci. USA 75, 1505-1509. SLISKI, A. H., ESSEX, M., MEYER, C., and TODARO, G. J. (1977). Feline oncornavirus-associated cell membrane antigen: Expression in transformed nonproducer mink cells. Science 196, 1336-1339. STEPHENSON, J. R., KHAN, A. S., SLISKI, A. H., and ESSEX, M. (1977). Feline oncornavirusassociated cell membrane antigen: Evidence for an immunologically cross-reactive feline sarcoma viruscoded protein. Proc. Nut. Acad. Sci. USA 74, 5608-5612. SVOBODA, J., POPOVIC, M., SAINEROVA, H., MACH, O., SHOYAB, M., and BALUDA, M. (1977). Incomplete viral genome in a non-virogenic mouse tumor cell line (RVP,) transformed by Prague strain of avian sarcoma virus. Int. J. Cancer 19, 851-858. TODARO, G. J., BENVENISTE, R. E., LEIBER, M. M., and LIVINGSTONE, D. M. (1973). Infectious type-C viruses released by normal cat embryo cells. Virology 55, 506-515. WONG-STAAL, F., GILLESPIE, D., and GALLO, R. C. (1976). Proviral sequences of baboon endogenous type-C RNA virus in DNA of human leukemia tissues. Nature (London) 262, 190-195. WONG-STAAL, F., REITZ, M. S., and GALLO, R. C. (1979). Retroviral sequences in a leukemic gibbon and its contact: Evidence for partial provirus in the nonleukemic gibbon. Proc. Nat. Acad. Sci. USA 76, 2032-2036.
99,
135- 144
Distribution R. KOSHY,” *Laboratory
(1979)
of Feline Leukemia Virus DNA Sequences Normal and Leukemic Domestic Cats F. WONG-STAAL,*
R. C. GALLO,“,’
W. HARDY,t
in Tissues
AND M. ESSEXS
of Tumor Cell Biology, National Cancer Institute, Bethesda, Maryland 20205; TMemorial Kettering Cancer Center, New York, New York 10021; and SDepartment of Microbiology, Harvard University School of Public Health, Boston, Massachusetts 02115 Accepted
July
of
Sloan-
16, 1979
Seroepidemiological studies indicate that leukemia and lymphoma in cats are horizontally transmitted and that feline leukemia virus (FeLV) is the agent responsible for the disease. Yet, in a significant proportion of leukemic cats, FeLV has not been identified, despite the presence of the feline oncornavirus-associated cell membrane antigen (FOCMA) and in some instances, epidemiological evidence indicating exposure to FeLV. Prompted by these so called virus negative cases of cat leukemia, we surveyed a total of 176 tissues from 50 cats for the presence of FeLV proviral DNA sequences. The animals studied included viremic and nonviremic cats that were either healthy or leukemic. We found: (1) DNA from virus-positive tissues from both healthy and leukemic cats hybridized 60-100% of lZ51-FeLV (strain Rickard) RNA. (These values are normalized to hybrid yield obtained with DNA of cat cells infected with FeLV in culture, which is 55%.) (2) A few virus-negative tissues also hybridized 60-100% of FeLV RNA. These tissues were derived from lymphomabearing cats as well as healthy cats. (3) The majority of virus-negative tissues hybridized 30-60% of FeLV RNA, and in this group, tissues from leukemic cats did not show significantly higher hybridization. (4) The results did not define a particular tissue target site since comparable hybridization results were obtained with many different tissues. (5) A few virus-negative tissues from healthy cats hybridized only 15-30% FeLV RNA. This is considerably lower than the level found with the majority of virus-negative tissues (normal or leukemic) and may reflect the true level of endogenous FeLV related sequences in cats. If so, the higher hybridization levels (30-60% of FeLV RNA) observed with most virusnegative tissues indicates more widespread infection in cats than previously believed, and suggests integration of only partial provirus and/or infection of only a fraction of the cells in the tissue. If exogenous FeLV is involved in the leukemogenesis of “virus-negative” cats, the apparent lack of detectable proviral sequences in tissues of many of these cats over and above those of healthy cats could be interpreted in different ways, e.g., partial loss of provirus, integration of a small fragment, or presence of proviral sequences in a limited population of cells. These “virus-negative” cats may serve as a model (S. M. Cotter and M. Essex, 1977, Amer. J. Pathol. 87,265-268) for other animals, including man, where leukemia may sometimes be associated with certain type-C related subviral markers, but detection of provirus is unusual. INTRODUCTION
are easily infected with FeLV when in the animals, and Many naturally occurring spontaneous company of virus-excreting when infected, they have a much higher risk leukemias and lymphomas in the domestic of developing leukemia and lymphomas cat (Felis catus) are caused by a type-C RNA tumor virus, feline leukemia virus (Hardy et cd., 1973b; Essex et aE., 1975, 1977a). The majority of infected cats with (FeLV), which is transmitted by horizontal leukemia and lymphomas have readily deinfection (Essex, 1975; Hardy et al., 1973; tectable free FeLV or viral antigens in their W. F. Jarrett et al., 1973). Uninfected cats tissues (Essex et al., 1975; Hardy et al.. 1973). There have be& some studies evalu: ating the presence of detectable FeLV re1 To whom reprint requests should be addressed. 135
0042-6822/79/150135-10$02.00/O Copyright All rights
0 1979 by Academic Press, Inc. of reproduction in any form reserved.
136
KOSHY
lated sequences in feline DNA. Gillespie, et al. (1973) demonstrated the presence of FeLV related sequences in the spleens of a few specific-pathogen-free normal cats. Quintrell et al. (1974) obtained about 30% hybridization of FeLV RNA to DNA from the livers of three normal cats which were negative for FeLV antigens. Okabe et al. (1976) showed FeLV related DNA in cat tissues including the livers of two specificpathogen-free cats. Benveniste et al. (1975) have reported that sequences partially related to the RNA of FeLV are present endogenously in all cats including specificpathogen-free animals. Levin et al. (1976) reported the presence of FeLV related sequences in six virus-negative tissues studied. Approximatley 30% of cats with spontaneous leukemia have neither free virus nor serologically assayable viral components (Hardy et al., 1969; Hardy, 1971; Essex, 1975). However, two lines of evidence suggest that FeLV might nonetheless be involved in the etiology of these virus-negative tumors: (a) The incidence of virus-negative cases of leukemia in cats that live in leukemia cluster households with virus-positive excretor cats is significantly higher than of those in carrier-free households (Essex et al., 1979); (b) feline oncornavirus associated cell-membrane antigen (FOCMA) which is a transformation-specific antigen known to be encoded by the related feline sarcoma viruses (FeSV) (Sliski et al., 1977; Stephenson et al., 1977; Sherr et al., 1978) is expressed in virus-negative leukemic cells (Hardy et al., 1977; Essex et al., 197713, 1979). It is this group of cats that we are particularly interested in because of possible similarities to other virus-negative species, especially man, where type-C virus related subviral molecules have sometimes been observed in association with leukemia (reviewed by Gallo and Meyskens, 1978). In naturally infected chickens (Neiman et al., 1975), cattle (Miller et al., 1976), and some primates (Scolnick et al., 1974; Gallo et al., 1978), it has been shown that tumor tissues contained retroviral sequences that normal tissues did not, suggesting transmission of acquired virus among these animals. Using DNA complementary (cDNA)
ET AL.
to subgroup B of FeLV (FeLV-B), Levin et al. (1976) observed extra copies of FeLV DNA in virus-positive tumor tissue that were not present in normal tissues, while no difference between normal and tumor tissue was detected in virus-negative cats. However, comparison was not made between normal and tumor tissues from the same cat. In this initial study we were interested in examining DNA from many tissues from each of a large number of cats for FeLV related sequences. Since we are studying field animals where the nature of the infecting agent may vary from one animal to the next, the choice of a particular subgroup of FeLV for probes appears arbitrary. However, of the three subgroups, FeLV-A is the most virulent strain, and is the most commonly occurring in nature. FeLV-A is always present in virus-positive cats with leukemia or lymphoma and can occur alone in a significant proportion of the cases, while FeLV-B or FeLV-C is always accompanied by FeLV-A, (Hardy et al., 1976; 0. Jarrett et al., 1978; Sarma and Log, 1973). Consequently, FeLV-A was used as probe. The results show that virus-positive cats almost invariably have greater amounts of hybridizable sequences than virusnegative cats, but with no apparent preferred target tissue for virus integration. However, there were no significant differences between normal and leukemic tissues in virus-positive or in virus-negative cats. Finally, DNA from a few virus-negative tissues from a few cats contained considerably lower levels of FeLV related sequences than all other samples. MATERIALS
AND
METHODS
Feline tissues. Tissues were obtained at autopsy from pet cats which had developed leukemidlymphoma, or from normal cats from the Animal Medical Center, New York; and the Angel1 Memorial Animal Hospital, Boston. Several specific-pathogen-free kittens were purchased from Liberty Laboratories, Liberty Corner, New Jersey. A total number of 176 tissues from 50 different cats were studied. The tissues were carefully examined by
FeLV
RELATED
SEQUENCES
light microscopy and in all cases there was no extensive infiltration of tumor cells in non-tumor tissues, and the tissues were not necrotic. Of the 50 cats, 14 were FeLV negative and healthy, 21 were FeLV negative and leukemic, 3 were FeLV positive and healthy, and 12 were FeLV positive and leukemic. The cats were classified as “virus positive” or “virus negative” by means of the immunofluorescence test (Hardy et al., 1973a,b) which detects expression of FeLV in the peripheral blood. Extraction of DNA. Fresh or frozen cat tissues were minced and homogenized in a Sorvall Omnimix homogenizer with 3 vol of 10 mM EDTA and 50 mM Tris, pH 9.0. The homogenates were treated with 1% sodium dodecyl sulfate (SDS, GallardSchlessinger) for 20 min at 60”. They were then extracted first with phenol:chloroform: isoamyl alcohol (1:1:0.2) and then with chloroform alone, precipitated with 2 vol of ethanol at -2O”, redissolved in 50 n-&f Tris (pH 9.0) and 0.5% SDS, and digested with Pronase (300 pug/ml). The DNA was reextracted as before and precipitated again with ethanol. DNA precipitates were dissolved in distilled water and sheared by sonication to an average size of 400 nucleotides. The sheared DNA was treated with 0.3 N NaOH for 18 hr at 37”, and then neutralized with sodium acetate (pH 4.5). Then, the DNA was precipitated with ethanol, collected by centrifugation in a RCB-B Sorvall centrifuge, and dissolved in water. Viruses. Subgroup A feline leukemia virus (FeLV), strain Rickard, was grown in feline embryo fibroblasts and concentrated from culture medium by centrifugation followed by equilibrium sedimentation in sucrose gradients. PurQication of viral RNA. FeLV was solubilized with 1% SDS in the presence of Pronase (300 gg/ml). RNA was extracted repeatedly with redistilled phenol (saturated with 50 m&f Tris pH 9.0) until the ratio of optical density at 260 rnp to that at 280 rnp was greater than 1.9. The RNA was then fractionated by ultracentrifugation on lo-30% glycerol gradients in a SW 40.1 rotor at 40,000 rpm for 3 hr at 4”. The ‘70 S RNA from the gradient was
IN DNA
OF CAT
TISSUES
137
precipitated with ethanol at -20” overnight and collected by centrifugation at 12,300 gin a Sorvall RCB-B centrifuge. Radioiodination of viral RNA was kindly performed by Dr. W. Prensky, Memorial Sloan-Kettering Cancer Center, New York. Following iodination, the RNA samples were precipitated with cetyltrimethylammonium bromide (Reitz et al., 1972). Precipitates were dissolved in 1 M sodium chloride and reprecipitated with ethanol at -20” overnight. The precipitates were collected by centrifugation and dissolved in 10 m.J4 Tris-HCl pH 7.0. Hybridization of nucleic acids. lz51-FeLV (Rickard) RNA, lo-20 pg (i.e., -1500 cpm), was added to 100 pg of cat DNA in a final volume of 12 ~1 containing 0.4 M sodium phosphate, pH 6.8. The mixtures were sealed in capillary pipets, boiled for 5 min, and incubated to a C,t of lo4 (uncorrected for [Na+]>. Hybrids were assayed as previously described (WongStaal et aZ., 1976) by RNase A digestion (50 pug/ml, 0.6 M NaCl, 1 hr, 37”). Percentage hybridization was calculated, and in each case, normalized to the value obtained by hybridizing lz51-FeLV RNA to DNA of tissue cultured cat cells infected with FeLV (55%). Repeat assays for several samples selected randomly yielded comparable values. All samples were received coded, and the diagnosis and viral status of the animals were known only after the hybridization values were obtained. Hybridization controls. Several samples from each range of hybridization were checked for ability to hybridize with lz51ribosomal RNA and lz51-RD114 RNA. Ribosomal RNA was a mixture of 28 and 18 S purified from HeLa cells and was a gift from Dr. W. Prensky. RD114 RNA was gradient purified 70 S RNA from virus grown in RD cells. The lz51-FeLV RNA used in the hybridizations were checked by hybridization to DNA obtained from livers of leopard cat (Felis bengalensis) and baboon (Papio cynocephalus). The leopard cat is a distant relative of the domestic cat, having cellular sequences in common with it, but not FeLV related sequences (Benveniste et al., 1975). Baboon DNA has endogenous viral sequences related to RD114 virus, an endog-
138
KOSHY
enous virus in the domestic cat (Benveniste et al., 1975). Neither leopard cat DNA nor baboon DNA hybridized more than 10% of lz51-FeLV RNA, thus indicating that the viral probe was reasonably free of contaminating feline cellular or RD114 viral sequences. Kinetics of hybridization. Hybridization reactions were set up as described above. Each DNA sample was incubated to different lengths of time (0 to 100 hr) to give C,t values of 0 to 104, respectively. The hybrids were analyzed as described previously (Wong-Staal et al., 1976) and the normalized values plotted. Thermal melting of hybrids. Hybridizations were done as described above. The hybrids were melted for 2 min each at the indicated temperature and assayed as before. A graph was plotted of the temperature of melting versus the percentage of hybrids remaining. The melting temperature (t,) was the temperature at which 50% of the RNA remained in hybrid form. RESULTS
Hybridization of Y-FeLV to Cat DNA
(Rickard)
ET AL.
between healthy and leukemic cats. Six tissues from a few nonviremic cats hybridized in the LOW range. These results and possible interpretations of them are detailed in the following. Virus-Positive Cats: Lack Target Tissue for Virus
of Preferred Replication
Hybridization values obtained with various tissues of individual viremic cats are presented in Fig, 1. DNA from almost all of the tissues, whether derived from healthy or leukemic cats, hybridized in the HIGH range. Surprisingly, neither the tumor tissues nor any other tissue hybridized FeLV RNA preferentially. These results indicate that there are no preferred target tissues, including the tumor tissues, for virus replication in the viremit cats. Although viral sequences seemed ubiquitous in viremic cats, one tissue appeared to be restrictive for virus replication. In four out of six cats in which DNA from muscle was examined, this DNA hybridized significantly less FeLV RNA (Fig. 1).
RNA
A total of 176 tissues from 50 cats were studied for FeLV related sequences in their DNA. Hybridization of lz51-FeLV RNA to DNA from leopard cat and from baboon was less than lo%, showing respectively that the FeLV RNA used as a probe was relatively free of contaminating feline cellular sequences as well as RD114 viral sequences. Four groups of cats were studied: nonviremic-healthy, nonviremic-leukemic, virem&healthy, and viremic-leukemic. For convenience of comparison between the groups, hybridization values are designated HIGH @O-100%), INTERMEDIATE (30SO%), and LOW (15-30%). DNA from most tissues of viremic cats hybridized in the HIGH range. Some tissues of nonviremic cats, both healthy and leukemic, also hybridized in the HIGH range, with no ap preciable difference in the levels between healthy and leukemic cats. The majority of tissues from nonviremic cats, however, hybridized in the INTERMEDIATE range, again with no significant differences
Virus-Negative
Cats
Leukemic versus healthy animals. DNA from a few tissues of virus-negative eats hybridized in the HIGH range. This was true for tissues from both healthy and leukemic cats (Figs. 2 and 3). DNA from the majority of tissues from virus negative cats, healthy or leukemic, hybridized in the INTERMEDIATE range. Thus, no increased amounts of proviral sequences were seen in tumor tissues of virus-negative cats compared to normal tissues of healthy virusnegative cats. On the basis of the results of this survey of multiple tissues from a large number of cats, it is unlikely that a nontarget (nontumor) tissue serves as a reservoir for virus integration and replication. Low hybridization values. A surprising result was that DNA from five tissues of four healthy cats and one tissue from a leukemic eat hybridized considerably lower amounts of FeLV RNA (l&30%) than DNA from all other cats (Figs. 2 and 3). These reactions were characterized by ki-
FeLV RELATED
SEQUENCES
IN DNA OF CAT TISSUES
139
FIG. 1. Hybridization of lZ51-FeLV-A (Rickard) RNA to DNA from virus-positive healthy and viruspositive leukemic cats. Various tissues from each cat (number N, or L,) were studied. Reactions were done in liquid at DNA excess, 0.4 M sodium phosphate and 60” to a C$ of lo4 (uncorrected for salt concentration). Values are normalized to that obtained by hybridizing FeLV-A RNA to DNA from feline cells infected with FeLV-A, and producing virus.2 DNA from control tissues (viz., from human, baboon, and leopard cat) hybridized FeLV-A RNA less than 1O%.2 Hybridization values are arbitrarily divided into three ranges, 10-30, 30-60, and 60-loo%.* N, normal cats; L, leukemic cat; BM, bone marrow; BR, brain; K, kidney; LI, liver; LN, lymph node; LU, lung; M, muscle; OM, omentum; S, spleen; SG, salivary gland; TF, thoracic fluid; TH, thymus; TO, tongue; TU, tumor.
netics and t, experiments as well (see following section). As an additional control, the quality of the DNA samples was checked by hybridization to labeled ribosomal RNA and RD114 viral RNA. The hybrid yields obtained for ribosomal RNA and for RD114 viral RNA were comparable with DNA samples from the LOW, INTERMEDIATE, or HIGH groups. It is, therefore, unlikely that the LOW values obtained for these samples with FeLV RNA were due to nonspecific inhibitors of hybridization such as contaminating nucleases in the DNA or due to degradation of the DNA. Characterization of the Hybrids by Kinetics and Therm& Stability Measurements
Division of the DNA samples into HIGH, INTERMEDIATE, and LOW ranges was substantiated by hybridization kinetics and thermal stability measurements of the hybrids formed. Figure 4 presents the results 2 Also applies to Figs. 2 and 3.
of kinetic experiments performed with DNA samples from the three different ranges, with DNA from feline cells (FEA) infected with FeLV-A as a positive control. The. results of these experiments corresponded closely with those of the first series of hybridization experiments. Samples from the different groups hybridized again as HIGH, INTERMEDIATE, or LOW. The results indicate that a complete provirus identical to FeLV-A is not present in DNA of all cat tissues as an endogenous virogene. A provirus at even one copy per cell hybridizes 80% of the viral RNA by a C$ of 104, with a C&2 of 5 x lo3 (Wong-Staal et al., 1976), which is more than the extents of hybridization obtained with DNA in either the LOW or INTERMEDIATE groups. Our data do not rule out the possibility of a few cells in a population containing complete FeLV-A provirus, or all cells containing a complete virus partially related to FeLV-A. Within each group, DNA from tumor or nontumor tissues hybridized with similar
140
KOSHY ET AL.
I N1
I N2
I, N3
I b
%
, N6
, NT
, NE
/ N9
Ii, NlD
, Nil
NV
N11
, NJ4
FIG. 2. Hybridization of lz51-FeLV-A (Rickard) RNA to DNA from virus-negative healthy cats. Reactions were carried out as described under Materials and Methods. BM, bone marrow; BR, brain; H, heart; K, kidney; LI, liver; LN, lymph node; LU, lung; M, muscle; S, spleen; SG, salivary gland; TH, thymus; TO, tongue.
kinetics, suggesting that there is no amplification of viral sequences in tumor tissues. Thermal stabilities of the hybrids formed between 1251-FeLV-A RNA and several DNA samples from each range of hybridization were also measured (Fig. 5). The t, values obtained correlated with the extents of hybridization. Thus, samples which hybridized HIGH had the same t, as
DNA from cat cells infected with FeLV-A (ht, = O”). The INTERMEDIATE samples displayed a lower t, (A&, = 29, and the LOW samples had the lowest t, (At, = 5”). DISCUSSION
A great deal of evidence indicates that FeLV is involved in the cause of naturally
FIG. 3. Hybridization of ‘7-FeLV-A (Rickard) RNA to DNA from virus-negative leukemic cats. The reactions were done as described under Materials and Methods. See Fig. 2 for symbol legends.
FeLV RELATED
SEQUENCES
occurring feline leukemia among virus-positive cats. However, approximately 30% of all leukemic cats are virus negative, and the role of FeLV, if any, in leukemia of these cats is not understood. Two separate lines of evidence suggest that leukemia in virus-negative cats may also be induced by FeLV: (a) Leukemic cells of these cats express FOCMA, a tumor-specific antigen induced by FeLV and FeSV (Hardy et al., 19’7’7; Essex et al., 1979; Sliski et al., 19’77). However, since radiation or chemically induced feline leukemia models have not yet been developed and since FeLV has not yet been directly shown to code for FOCMA! this observation does not exclude the possibility that FOCMA is a cell antigen induced by FeLV, and might also be activated by nonviral leukemogens. (b) The incidence of leukemia in virus-negative cats known to be exposed to FeLV is as high as that among exposed cats which become viremic. This increase is greater than would be expected due to chance alone (Essex et al., 1978). Several studies have been conducted to evaluate the presence of FeLV provirus sequences in healthy and leukemic cats (Gillespie et al., 1973; Quintrell et aE., 1974; Okabe et al., 1976; Benveniste et al., 1975; Levin et al., 1976). However, these only
FIG. 4. Kinetics of hybridization of lZ51-FeLV-A RNA to DNA from various cat tissues. Reactions were done as described under Materials and Methods. x - - - x Feline embryo cells infected with FeLV-A. l AOvm Tissues from INTERMEDIATE hybridization group. OV Tissues from INTERMEDIATE hybridization group. DA Tissues from LOW hybridization group.
141
IN DNA OF CAT TISSUES
80 P ?._ E d ; g-? 0
60
40
20
0
60
70
80 Temperature
90
100
IQ
FIG. 5. Thermal melting profiles of hybrids of lZ51-FeLV-A RNA and cat cellular DNA. Hybrids were assayed as described under Materials and Methods. Profiles shown are representative of several samples with “HIGH,” “INTERMEDIATE,” and “LOW” hybridization, respectively. + Feline embryo cells infected with FeLV-A; t, = 82”. n HIGH hybridization; t, = 82”. A INTERMEDIATE hybridization; t, = 80”. 0 LOW hybridization. t, = 77”.
surveyed a small number of samples and usually included only tumor tissues from the leukemic cats. The interpretations were further complicated by the difficulty in distinguishing between endogenous and acquired FeLV related sequences in DNA of cats. In order to understand better the roles of FeLV in leukemogenesis in both viremic and nonviremic cats, and to compare feline leukemia with spontaneously occurring, viral-induced leukemias of other species, we examined DNA from multiple tissues from a large number of cats. These included virus positive-healthy and virus positive-leukemic, virus negative-healthy and virus negative-leukemic cats. The most commonly isolated viruses from cats with lymphomas belong either to subgroup A (FeLV-A) or are mixtures of viruses from subgroup A and subgroup B (FeLV-AB). Subgroup C (FeLV C) is rare and occurs only in mixtures with FeLV A (FeLV-AC) or with FeLV-AB (FeLV-ABC) (Hardy et al., 1976; W. Jarrett et al., 1973; Sarma and Log, 1973). Consequently, RNA from the Rickard strain of FeLV, a virulent strain (Rickard et al., 1969) comprising predominantly subgroup A, was used as the probe.
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The results yielded several interesting observations: (1) The presence of proviral DNA sequences is widespread among the tissues of virus-positive cats, healthy or leukemic, indicating a lack of preferred target tissue(s) for the virus. Among leukemic cats, provirus was detected in almost every tissue in addition to the tumor tissue. This result cannot be due to infiltration of leukemic cells into non-tumor tissues since (a) no extensive infiltration was observed by microscopic examination of the tissues, (b) similar rates of hybridization were obtained with tumor or non-tumor tissues, and (c) in some cases, the DNA from tumor tissues hybridized even less probe than DNA from non-tumor tissues. Similar results have been reported in a case of naturally occurring gibbon ape leukemia, in which proviral sequences of gibbon ape leukemia virus (GaLV) were detected to the same extent in all tissues of the viremic animal except muscle and brain (Gallo et al., 1978). The extent of virus spread may correlate with the level of virus production since both cats and gibbons are high virus excretors. (2) In four out of six viremic animals in which the muscle was examined, DNA from this tissue hybridized considerably lower amounts of FeLV RNA, suggesting that this tissue may be restrictive for virus replication. In the few instances where the muscle contained levels of proviral sequences comparable to those of other tissues, infection may have occurred congenitally. Again the restriction of virus replication in muscle has been observed in viremic gibbons infected postnatally (Gallo et al., 1978; Kawakami et al., 1978). (3) While the majority of virus-negative cat tissues hybridized in the INTERMEDIATE range, DNA from tissues of some “virus-negative” cats (both healthy and leukemic) hybridized the same amount of viral RNA as did virus-positive cats (i.e., 60-100%) (Fig. 2). This extensive hybridization clearly shows that at least in a fraction of the so-called virus-negative cats FeLV infection did take place, even though serological tests failed to indicate this. (4) DNA from some tissues of a small
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number of virus-negative cats hybridized unusually LOW amounts of FeLV RNA (15-30%) (Figs. 2 and 3). These results were reproducible and could not be due to degradation of the DNA samples since these DNA samples hybridized ribosomal RNA and RD114 RNA as efficiently as DNA from tissues in the HIGH and INTERMEDIATE range. There are two possible interpretations of these results, which are not mutually exclusive. First, since cats are an outbred species, there may be a wide variation of copy number and divergent genetic loci of the endogenous FeLVrelated virogenes from animal to animal. Second, the LOW values may be the real endogenous FeLV related sequences and the INTERMEDIATE DNA samples contain endogenous as well as novel viral sequences acquired by infection. Results of hybrid analysis by t, determination tend to support the second possibility. Since the LOW level hybrids have the lowest t, values (At, = 5”) the data indicate a divergence of sequences as would be expected for endogenous FeLV related sequences. The INTERMEDIATE level hybrids exhibit t, values intermediate between the LOW and HIGH hybridization samples, consistent with presence of partly endogenous and partly recently acquired sequences. If this hypothesis is correct, then there must be much more widespread infection of cats by FeLV than previously believed since DNA from the majority of virus-negative cats hybridized more viral RNA (INTERMEDIATE range). The lack of complete hybridization in these virus-negative cats could mean that either only part of the provirus is present in the host DNA (due to integration of partial provirus or due to loss of some sequences after integration), that proviral sequences are integrated into the DNA of only a small number of cells, or both. Until recently there was little evidence for incomplete proviral sequences in cells transformed by type-C retroviruses. Svoboda et al. (1977) detected incomplete viral genomes in a mouse nonproducer cell line which had been transformed by Rous sarcoma virus (RSV): Hybridization of the cellular DNA with RSV 35 S RNA yielded a lower plateau
FeLV
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SEQUENCES
value than was obtained with DNA from a virogenic line. Recently, by means of restriction endonucleases and “blot” hybridization experiments, Wong-Staal et al. (1979) provided direct evidence for partial provirus in DNA from the liver and kidney of a gibbon ape which had been exposed to GaLV. There may be subtle differences between the leukemic and normal virus-negative eats, which cannot be discerned by means of the hybridization techniques used. These differences may become more apparent upon analysis with restriction endonucleases and hybridization of viral probes to specific DNA fragments. Such experiments are now in progress. ACKNOWLEDGMENTS This work was partly supported by NC1 Grants CA13885, CA-16599, CA-18488 and by American Cancer Society Grant DT-32. R.K. is a Fellow of the Fogarty International Center, NIH. W.H. is a scholar of the Leukemia Society of America. REFERENCES BENVENISTE, R. E., SHERR, C. J., ~~~TODARO, G. J. (1975). Evolution of type-C viral genes: Origin of feline leukemia virus. Science 190, 886-888. COWER, S. M., and ESSEX, M. (1977). Animal model: Feline acute lymphoblastic leukemia and aplastic anemia. Amer. J. Pathol. 87, 265-268. ESSEX, M. (1975). Horizontally and vertically transmitted oncornaviruses of cats. Advan. Cancer Res. 21, 175-246. ESSEX, M., COTTER, S. M., HARDY, W. D., HESS, P., JARRETT, W., JARRETT, O., MACKEY, L., LAIRD, H., PERRYMAN, L., OLSEN, R. G., and YOHN, D. S. (1975). Feline oncornavirus-associated cell membrane antigen. IV. Antibody titers in cats with naturally occurring leukemia, lymphoma, and other diseases. J. Nat. Cancer Inst. 55, 463-467. ESSEX, M., CORER, S. M., SLISKI, A. H., HARDY, W. D., STEPHENSON, J. R., AARONSON, S. A., and JARRETT, 0. (1977a). Horizontal transmission of feline leukemia virus under natural conditionsIn a feline leukemia cluster household. Znt. J. Cancer 19, 90-96. ESSEX, M., GRANT, C. K., COTTER, S. M., SLISKI, A. H., and HARDY, W. D. (1979). Leukemia specific antigens: FOCMA and immunosurveillantie. In “Modern Trends in Human Leukemia” (R. Neth, R. C. Gallo, P. H. Hofschneider, and K. Mannweiler, eds.), Pt. III. Springer-Vex-lag, New York, pp. 453-467. ESSEX, M., SLISKI, A. H., HARDY, W. D., DENOR-
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