Rat Sequences of the Kirsten and Harvey Murine Sarcoma

Transcription

1 JOURNAL OF VIROLOGY, Feb. 1976, p Copyright American Society for Microbiology Vol. 17, No. 2 Printed in U.S.A. Rat Sequences of the Kirsten and Harvey Murine Sarcoma Virus Genomes: Nature, Origin, and Expression in Rat Tumor RNA GARTH R. ANDERSON'* AND KEITH C. ROBBINS Viral Leukemia and Lymphoma Branch, National Cancer Institute, Bethesda, Maryland 20014, and Hazleton Laboratories America, Inc., Vienna, Virginia Received for publication 6 August 1975 Two murine sarcoma viruses, the Kirsten and the Harvey, were isolated by passage of mouse type C leukemia viruses through rats. These sarcoma viruses have genomes containing portions of their parental type C mouse leukemia virus genomes, in stable association with specific rat cellular sequences that we find to be quite likely not those of a rat type C leukemia virus. To determine if these murine sarcoma viruses provide a model relevant to the events occurring in spontaneous tumors, we have hybridized DNA and RNA prepared from rat tumors and normal rat tissues to [3HIDNA prepared from the Kirsten murine sarcoma virus. We have also hybridized these rat tissue nucleic acids to [3H ]DNA prepared from a representative endogenous rat type C leukemia virus, the WFU (Wistar-Furth). Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected in the DNA of both tumor and normal tissues, with no evidence of either gene amplification or additional sequences being present in tumor DNA. Sarcoma-viral rat cellular sequences and endogenous rat leukemia viral sequences were detected at elevated concentrations in the RNA of many rat tumors and in specific groups of normal tissues. Endogenous type C leukemia viruses have been isolated from numerous species (14, 40). They appear to be vertically transmitted, residing in a latent form within cellular DNA (4, 9, 36). Both laboratory strains and endogenous isolates of type C leukemia viruses have been shown to induce leukemia in vivo after long latent periods (13, 40). The role, if any, that the endogenous type C viruses may play in spontaneous and carcinogen-induced tumors is not clear. Type C sarcoma viruses have been isolated as apparent derivatives of the leukemia viruses. In contrast to the leukemia viruses, sarcoma viruses rapidly induce solid tumors in vivo and transform cells in culture (40). Sarcoma virus genomes in both murine and avian systems have been found to contain partial or complete sets of leukemia virus genes (5, 11, 33, 35, 37), along with additional nucleic acid sequences (29, 35). Kirsten murine sarcoma virus (KiMSV) and Harvey murine sarcoma virus (HaMSV), which arose after passage of the Kirsten murine leukemia virus (KiMuLV) and Moloney murine leukemia virus (M-MuLV) I Present address: Department of Microbiology, University of Pittsburgh, School of Medicine, Pittsburgh, Pa through rats (15, 21), each have genomes containing portions of their parental mouse leukemia virus genomes (5, 35), along with specific rat sequences (31, 37, 42). Hybridization seen with KiMSV- and HaMSV-infected cell RNA to the reverse transcriptase DNA product of a rat leukemia virus produced by normal rat kidney (NRK) cells has led to the assumption that the rat sequences in these two sarcoma virus genomes are those of a type C rat leukemia virus (24, 37, 42). Although Scolnick et al. (34) have raised the possibility that these rat sequences may be nonviral, a more recent report has returned to the hypothesis that these rat sequences are those of a rat leukemia virus (24). We present here results extending findings of Scolnick et al. (34) and Tsuchida et al. (42), which make it unlikely that the rat sequences in these rat-derived sarcoma virus genomes are those of a rat type C leukemia virus, but instead are other cellular sequences. According to the oncogene theory of Huebner and Todaro (16), increased expression of an endogenous type C leukemia viral gene is somehow responsible for most spontaneous and carcinogen-induced tumors. Martin and Weiss (27) have suggested an alternate theory that seems 335

2 336 ANDERSON AND ROBBINS readily applicable to the Kirsten and Harvey sarcoma viruses, where recombination of endogenous leukemia viral genes with a crucial set of other specific cellular genes leads to a transforming activity. By both theories, in the rat model system one would expect to find in tumors enhanced expression of some endogenous rat type C leukemia viral genes. By the second model, those rat cellular sequences that became a part of the Kirsten and Harvey murine sarcoma virus genomes should also be expressed at elevated levels in rat tumors. A [3H]DNA product synthesized by KiMSV, grown with helper KiMuLV in NRK rat cells, was used as an easily available hybridization probe to quantitate the presence of those rat cellular sequences (murine sarcoma virus [MSV]-rat sequences) present in the KiMSV and HaMSV genomes. The Wistar-Furth (WFU) rat leukemia virus was used as a source of [3H ]DNA to probe for type C rat leukemia viral nucleic acid sequences. Through hybridization to rat cellular DNA, we have confirmed the presence of endogenous leukemia viral sequences and murine sarcoma virus-specific rat cellular sequences within the DNA of normal and tutored rat cells. Postulating that the controlling factor in such gene expression is at the transcriptional level, we have further examined a series of RNAs prepared from 22 rat tumors and from more than 30 normal rat tissues. MATERIALS AND METHODS Cells. Cells were grown in the Dulbecco modification of' Eagle medium supplemented with 10%f calf serum (Colorado Serum Co., Denver, Colo.). The derivation of continuous lines of BALB/3T3 (BALB) and NIH/3T3 (NIH) mouse cells (18), and Osborne- Mendel (O-M)-derived NRK (10) has been reported. Two N.RK clones were used: a control clone showing no evidence of virus production, as measured by supernatant reverse transcriptase activity and by electron microscopy examination (kindly performed bv VT. Zeve, National Institutes of Health), and a second NRK clone that was spontaneously producing type C virus. The development of' KiMSV-transformed nonproducer clones of NIH (K-NIH). NRK (K-NRK), and BALB (K-BALB) has been described (3). Rat virus-producing Jones chloroma cells were a gift from J. Greenberger, Harvard Medical School. Viruses. The viruses used included clonal strains of KiMSV (KiMuLV) grown in NRK or Fischer rat cells to obtain a high ratio of sarcoma to helper leukemia virus (23), and KiMuLV grown in NRK rat or NIH mouse cells (3). Rat leukemia viruses (RaLV), designated by the strain from which each was isolated, included RaLV spontaneously produced by NRK cells (OM-RaLV), an isolate from a stock of MSV-O (Fischer-RaLV) (1), and two others, WFU- J. VIROL. RaLV (from Wistar-Furth) and WR-9-RaLV (from ACI), generously provided as virus-producing rat cell lines by P. Sarma, National Cancer Institute, and R. Peters. Microbiological Associates, Walkersville, Md. Harvey sarcoma virus was a generous gift from H. Temin, University of VWisconsin. Rat tumors. Transplanted rat tumors were obtained from P. Pluka at the Mason Research Institute, and from N. Greenberg and R. Herberman of' the National Institutes of Health. The primary spontaneous Fischer rat leukemia was obtained from W. Moloney, Harvard Medical School, and the dimethylbenzanthracene-induced primary tumors were obtained from J. Everly, Hazleton Laboratories. Three tumor-derived cell lines were obtained from R. Peters, National Institutes of Health. Wistar-Furth (W/FU). Osborne-Mendel (O-M), F-344 Fischer, and Sprague-Dawley rats were obtained from the National Institutes of' Health. Molecular hybridization. [3H ]DNA products were prepared by the endogenous reverse transcription reaction of' viruses purified by isopycnic centrifugation. Single-stranded [3HIDNA, labeled with all four deoxynucleoside triphosphates, was synthesized during a 6-h incubation at 37 C in the presence of 20 mg of actinomycin D per ml (25). Such DNAs synthesized in a reaction mixture also containing unlabeled deoxynucleoside triphosphates at 10-4M had specific activities of 3 x 103 counts/min per ng. Cellular and viral RNA were prepared by the hot phenol method (32), and cellular DNA was prepared by the method of' Marmur (26). Hybridization was performed in 38% formamide, 1 mm EDTA, 15 mm Tris (ph 7.5), and 150 mm NaCl, in a volume of 0.05 ml. Incubation was at 43 C for 7 days. Cellular RNA or DNA in the range of 0.1 to 2,000 jig was annealed to 0.4 to 1.0 ng of' [3H]DNA product. Hybridization was assayed by the S1 nuclease method (5, 22). Input counts were defined as those trichloroacetic acid precipitable in the absence of incubation and SI nuclease digestion. The background was taken as the number of SI nucleaseresistant [3H]DNA counts at zero incubation time and was uniformly less than 2%( of the input counts. Control tubes containing the highest concentrations of RNA were also assayed at zero time to monitor for RNA inhibition of S1 nuclease; these were never significantly inhibitory (<3%c). In reconstruction studies, the amounts of cellular RNA used did not interfere with virus-specific hybridization. 70S viral RNA hybridized 50% of' the homologous viral [3H ]DNA product at RNA:DNA molar ratios of' 2:1 to 5:1. At RNA:DNA ratios of greater than 10:1, 90 to 100% of each DNA probe was hybridized. Under the conditions of Stephenson and Aaronson (38), where incubation is at 60 C in 0.1 M K2HPO4 and hybridization is monitored by RNase A resistance, viral DNA product hybridized 50% of the homologous 32P-labeled 70S viral RNA at DNA:RNA molar ratios in the range of 2:1 to 5:1, and required a ratio of at least 20:1 to achieve 90%' hybridization. Recycling viral DNA product. DNA probes specific for particular RNAs were obtained by annealing viral DNA product with an excess of' the particular RNA for 18 h. S1 nuclease was then used to digest the

3 VOL. 17, (20 VIRUS RNA IN RAT TUMORS 337 nonhybridized DNA. After phenol and ether extractions, the surviving [3H IDNA was incubated in 0.5 N NaOH overnight at 43 C, neutralized with acetic acid, and dialyzed first against 0.1 M NaCl, 0.01 M Tris (ph 7.4), M EDTA, and then water. After lyophilization, the ['H ]DNA was resuspended in 0.01 M phosphate (ph 6.8). Recovery of initially hybridized DNA averaged approximately 80%. Such preannealing and processing caused small but significant loss of ability of the surviving DNA to hybridize. Control studies with homologous virus-producing cell RNA to hybridize viral DNA product showed that, in an initial annealing, 95 to 100% of the DNA would hybridize. In contrast, after preannealing, only 75 to 85% of this recycled DNA would hybridize with homologous RNA. Hybridization to tissue nucleic acids. In analysis of tissue nucleic acid, hybridization to WFU-RaLV [IH IDNA is presented directly as the percentage hybridized. With the KiMSV genome being composed of sequences of KiMuLV and of rat origin, hybridization of rat tissue RNA to KiMSV (KiMuLV) ['H ]DNA was normalized to the rat-specific fraction of this probe, as determined by the saturation level of hybridization (40%) given with NRK RNA. To ensure that only the MSV-rat sequences were being detected by this probe, control hybridization of tumor RNA with [3H]DNA prepared from NIH-grown KiMuLV showed all had less than 3% homology to the mouse leukemia virus probe. As a further confirmation that only MSV-rat sequences were detected, representative tumor RNAs were also hybridized to the rat-specific fraction of KiMSV (KiMuLV) ['H IDNA prepared by preannealing to NRK cellular RNA. These preannealed probes gave results indistinguishable from the normalized results obtained with the non-preannealed probe. Representative RNAs were also hybridized to a ['H ]DNA probe specific for all KiMSV sequences, which was prepared by preannealing [3H ]DNA product of KiMSV (KiMuLV) with RNA from KiMSVtransformed nonproducer NIH cells. These hybridizations gave results very similar to the results obtained with the KiMSV (KiMuLV) [3H ]DNA directly. To preclude the possibility that DNA was contaminating RNA preparations, each RNA was shown to lose all ability to hybridize after overnight treatment in 0.5 N KOH. To confirm the significance of negative results, each tissue RNA was found not to inhibit hybridization of Rauscher MuLV ['H]DNA with Rauscher viral RNA. RESULTS Mammalian type C sarcoma viruses replicate only in the presence of helper leukemia virus (2). Biochemical analysis of a sarcoma viral genome requires sources of the sarcoma virus nearly free of helper leukemia virus. Two techniques make such analysis of the Kirsten sarcoma virus feasible. First, KiMSV grown with helper leukemia virus in NRK rat cells yields virus stocks that have a high ratio of KiMSV to their helper virus (23, 31). Second, transformed nonproducer cell lines have been isolated that are infected with only sarcoma virus (2). Rat sequences in the nucleic acid of type C viruses grown in rat cells. Utilization of sarcoma virus stocks grown in the NRK cell line for genomic analysis is complicated by the phenomenon that type C viruses grown in rat cells can package rat cellular RNA sequences (34). This is not genetic recombination since such sequences do not transmit with virus infection. As illustrated in Fig. 1A, NRK cellular RNA hybridized 40% of the [3H ]DNA product prepared from KiMuLV grown in NRK cells, whereas NIH mouse cell RNA hybridized r (A) _ (B) I I~~~~~~~~~~~~~ IU 0 ION ~~ I-... I~... so /:- o R80~~5 10 1I I I~~~~~ RNA CONCENTRATION (,~g/50oul) FIG. 1. Rat sequences in the reverse transcriptase [3H JDNA product of NRK-grown virus. Hybridization to cellular RNA was under conditions described in the text. (A) Hybridization to [3H JDNA product of KiMuLV grown in NRK cells; (B) hybridization to [SHJDNA product of KiMuLV grown in NIH cells. The cellular RNAs used were: KiMuLV-NRK (U); NIH cells infected with KiMuL Vgrown in NRK (A), NIH cells infected with KiMuLV grown in NIH (0); uninfected, virus-nonproducing NRK (0); uninfected, virus-nonproducing NIH (0).

4 338 ANDERSON AND ROBBINS less than 5% of this DNA product. Correspondingly, only 60% of this ['H]DNA is hybridized by RNA from NRK infected with KiMuLV. In contrast, both NRK and NIH celp RNA showed less than 5% homology to the [3H ]DNA product of KiMuLV grown in NIH, and both NRK-producing KiMuLV and NIH-producing KiMuLV cell RNA hybridized over 95% of this DNA product (Fig. 1B). Growth in NRK cells can clearly result in the packaging by type C viruses of large amounts of cellular sequences, whereas growth in NIH cells apparently does not. The rat sequences present in the NRK-grown Ki- MuLV DNA product cannot represent a stable portion of the viral genome, since passage of NRK-grown KiMuLV to NIH cells resulted in transmission of KiMuLV but not NRK sequences. To determine if such results were specific either to the NRK cell line or to KiMuLV, the Fischer rat cell line and other type C viruses were also examined. As shown in Table 1, the packaging of rat sequences was seen with both BALB:virus-2 and Woolly monkey leukemia virus, grown in both NRK and Fischer rat cells. The extent of rat cellular sequences packaged was as high as 76% of the DNA product of BALB:virus-2 grown in NRK. Approximately half of the 70S RNA prepared from KiMSV (KiMuLV) grown in NRK cells is represented by rat sequences packaged by Ki- MuLV grown in NRK. Such 70S RNA labeled with 32P was hybridized to the DNA product of NIH-grown KiMuLV, NRK-grown KiMuLV, TABLE 1. Rat cellular sequences in the DNA products of rat-grown viruses Hybridization (%) with cellular RNA from: [3H ]DNA prod- Grown in: BALB:V- WLV ucts of virus 2-infected infected NRK ]_NRK NRK BALB:V-2 NRK Fischer rat Human 96 2 Woolly monkey NRK leukemia vi- Human 94 2 rus (WLV) J I a Hybridization of cellular RNA to viral DNA product was for 7 days at 43 C in a 0.05-ml reaction mixture composed of 38%9 formamide, 1 mm EDTA, 15 mm Tris, ph 7.5, 150 mm NaCI, 0.3 ng of ['H]DNA, and 20 to 400 fig of cell RNA. Hybridization was assayed with nuclease S1. Saturating levels of hybridization are given and represent the average of three points. J. VIROL. and NRK-grown KiMSV (KiMuLV) (Fig. 2). While the DNA product of NIH-grown KiMuLV hybridized only 20 to 25% of this RNA, the DNA products of both KiMuLV alone and KiMSV (KiMuLV) grown in NRK hybridized 55 to 60% of this RNA. Defining a virus genome as its 70S RNA, the rat sequences present in the NRKgrown KiMuLV DNA product must represent nearly all the differences between KiMuLV grown in NIH cells and KiMSV (KiMuLV) grown in NRK. The additional KiMSV sequences reported by Scolnick et al. (33) may either not be in 70S viral RNA or be an artifact of nonrepresentivity of DNA products. Relationship of packaged sequences to those rat sequences in the KiMSV genome. Comparison of those rat sequences packaged and reverse transcribed to DNA by type C virus grown in NRK cells with those rat sequences present in KiMSV shows that only about half of the packageable rat sequences are a stable portion of the KiMSV genome (Table 2), as defined by their passage with KiMSV. By preannealing to NRK cell RNA and digestion of nonhybridized [3H]DNA with S1 nuclease, the rat-rna homologous fractions of the DNA products of NRK-grown KiMuLV and KiMSV H DNA CPM X 10-3 FIG. 2. Hybridization of 1 ng of 32P-labeled 70S viral RNA of KiMSV (KiMuLVI grown in NRK with the 3H-labeled reverse transcriptase DNA products of the viruses: KiMuLV grown in NIH (0), KiMuLV grown in NRK (0) and NRK-grown KiMSV (Ki- MuLV) (A). Annealing was for 5 days at 60 C in 0.12 ml of 0.1 M KHPO4, ph 6.8. Hybridization was assayed by the resistance of the [32P]RNA to degradation by RNase A (38). The specific activity of each DNA product was 3 x 103 counts/min per ng. The biological ratio of KiMSV to KiMuLV in the KiMSV stocks was greater than 8:1.

5 VOL. 17, 1976 TABLE 2. Common rat sequences in KiMSV and HaMSVa Determinants Hybridization (%) to NRK-RNA preannealed fraction of [3H ]DNA product of NRK grown (KiMuLV) KiMuLV Cellular RNA Uninfected NRK BALB 4 15 NIH 6 12 KiMSV infected KiMSV (KiMuLV)-NIH K-BALB K-NIH HaMSV infected HaMSV (M-MuLV)-NIH HaMSV (M-MuLV)-NIH NTc 39 + K-NIH M-MuLV infected M-MuLV-NIH 8 14 Viral RNA KiMSV (KiMuLV)-NRK NT 76 KiMuLV-NRK NT 78 KiMuLV-NRK (digested NT 0 with alkali) KiMuLV-NIH 6 10 Cellular DNA NRK (rat) 52 NT NIH (mouse) 3 NT a ['HIDNA was prepared via the endogenous reaction from NRK-grown KiMuLV and KiMSV (Ki- MuLV). Each [3H]DNA was prehybridized to NRK cellular RNA (2 mg/ml), as described in the text. Forty-two percent of the KiMuLV [3H ]DNA and 38% of the KiMSV (KiMuLV) [3H ]DNA was hybridized in this preannealing. These rat-specific ['H]DNA fractions were then hybridized to RNA or DNA under the standard conditions described. Saturating levels of hybridization are given, and represent the average of three points (cellular RNA, 100 to 300 Mg; viral RNA, 0.5 to 2 jg; and cell DNA, 50 to 100 Mg). b M-MuLV, Moloney murine leukemia virus. c NT, Not tested. (KiMuLV) were isolated. RNA from KiMSVinfected NIH and KiMSV-infected BALB cells hybridized around half of the NRK RNAspecific fraction of the [3H ]DNA prepared from NRK-grown KiMuLV, but hybridized nearly completely the NRK RNA-specific fraction of [3H]DNA prepared from NRK-grown KiMSV VIRUS RNA IN RAT TUMORS 339 (KiMuLV). NIH and BALB cellular RNA showed less than 15% homology to either DNA. The fact that KiMSV nonproducer cell RNA nearly completely hybridizes the rat-specific fraction of the KiMSV (KiMuLV) [3H ]DNA further indicates that either KiMSV (KiMuLV) contains very few loosely associated packaged rat sequences or can no longer reverse transcribe them. Harvey sarcoma virus-infected NIH cells and KiMSV-infected NIH cells contain the same limited proportion of those rat sequences packaged by type C viruses grown in NRK cells, since HaMSV (M-MuLV)-infected NIH cell RNA and K-NIH cell RNA both hybridized to the same level (40%) of the NRK-specific fraction of KiMuLV-NRK DNA, and these in fact represent the same sequences, since mixing of the two gave no additivity (Table 2). RNA from NIH mouse cells infected with HaMSV also hybridized to nearly all the rat-specific DNA prepared from KiMSV (KiMuLV) grown in NRK. Since alkali digestion destroyed the ability of NRK-grown KiMuLV viral RNA to hybridize to either rat-specific DNA, the rat sequences detected in this viral RNA must truly represent RNA and not DNA packaged by the virions. Confirmation that the KiMSV sequences seen in NRK cellular RNA are of rat origin was seen by their presence in NRK rat cellular DNA and lack of delectability in NIH mouse DNA (Table 2). All together, these results show that growth of type C virus in the NRK cell line results in packaging of specific cellular RNA, which can function as a template for the viral reverse transcriptase. Around half of this RNA is propagated with KiMSV and HaMSV as an integral part of the virus genome. Absence of homology with rat type C viruses. To determine if those rat sequences packaged and reverse transcribed by type C virus grown in NRK cells may be those of a rat type C virus, we first examined the homology of KiMSV with a type C leukemia virus produced by Wistar-Furth rats, the WFU-RaLV. Figure 3 illustrates the complete lack of homology between the WFU-RaLV and KiMSV (KiMuLV) ['HIDNA probes. Homologous viral RNA completely hybridized each DNA, with a Crts of 0.15 mol x liter/s for WFU-RaLV and 0.4 mol x liter/s for the KiMSV. At exceedingly high Crt values, cross-hybridization was observed, presumably reflecting packaging of the heterologous rat RNAs (6). Four additional isolates of RaLV were also examined (Table 3). RNA from each of the five

6 340 ANDERSON AND ROBBINS z 80 L&J 0~~~~~~ // 60/ -40- X-20 0;/ g 0~~~~~~~ lo1 102 Crt (mole x sec/liter) FIG. 3. The absence of relatedness between WFU- RaLV and KiMSV (KiMuLV). Each virus RNA was hybridized to around 0.5 ng of the homologous and heterologous [3HJDNA products. Hybridization was for 7 days at 43 C in a 0.05-ml reaction volume composed of 38% formamide, 0.15 M NaCI, 0.01 M Tris (ph 7.5), and M EDTA, with the Si nuclease method being used to monitor hybridization. Symbols: WFU-RaLV viral RNA hybridized to WFU- RaLV [3H]DNA (0), WFU-RaLV viral RNA hybridized to KiMSV (KiMuLV) [3H JDNA (U), KiMSV (KiMuLV) viral RNA hybridized to WFU-RaLV [3HJDNA (0), and KiMSV (KiMuLV) viral RNA hybridized to KiMSV (KiMuLV) [3H JDNA (0). isolates of RaLV hybridized to more than 85% of ['H ]DNA prepared from the WFU RaLV virus. In contrast, KiMSV (KiMuLV)-infected cell RNA showed less than 6% homology to the same RaLV DNA probe. Consistent with this, hybridization to [9H ]DNA prepared from NRK-grown KiMSV (KiMuLV) was less than 4% for three of the five RaLV isolates. RNA from Jones chloroma virus-producing rat tumor tissue gave 11% hybridization of the KiMSV DNA. However, many natural rat tumors express elevated levels of KiMSV-specific rat RNA sequences (see below). In marked contrast to the other RaLV producers, however, OM-RaLV cellular RNA hybridized 35% of the KiMSV (KiMuLV) DNA. This RNA is from NRK cells (originally derived from an O-M rat) spontaneously producing RaLV. It was important to determine if the KiMSVhomologous RNA seen in our OM-RaLV-producing NRK cells truly reflects that of an endogenous type C virus, since (i) NRK RNA alone shows substantial homology to KiMSV ['H]DNA (34, 42), and (ii) there are reports of homology between KiMSV and an NRK-RaLV (24, 37). RNA from normal, virus-negative NRK cells was thus compared to that from the OM-RaLV-producing clone. As shown in Fig. 4, virus production had no effect on the concentration of KiMSV-homologous RNA in the NRK cells. In contrast, the RaLV-producing NRK cells had at least a 50-fold greater concentration of WFU-RaLV-homologous sequences. These findings, similar to those obtained by Scolnick et al. (34), demonstrate that the homology to KiMSV [3H ]DNA seen with OM-RaLV-producing NRK cells cannot be taken to indicate partial homology of this particular RaLV with KiMSV, as it merely reflects that homology shown by NRK alone. The 35S subunit size of type C viral RNA provides an additional (albeit nonrigorous) criterion for type C leukemia virus (40), although 20S and 30S RNA species homologous to sarcoma viruses have been seen intracellularly in MSV-producing cells (44, 45). The rat sequences rescued by KiMuLV grown in NRK TABLE 3. J. VIROL. Absence of homology between RaLV isolates and KiMSVa Hybridization (%) to WFU-RaLV KiMSV Type C producer [3H -NA (KiMuLV) ['H JDNA Cellular RNA (100 Mg) WFU-RaLV Fischer-RaLV 97 3 WR-9-RaLV 98 2 OM-RaLV Jones chloroma RaLV (10 4g) KiMSV (KiMuLV)-NRK 4 97 Viral RNA (100 ng) WFU-RaLV KiMSV (KiMuLV)-NRK 5 95 Control cellular RNA (100 Mg) Fischer NRK 9 36 OMembryo 7 6 a RNA concentrations sufficient to saturate homologous viral ['HJDNA probes were hybridized under standard conditions described in the footnote to Table 1. It should be noted that massively higher RNA levels gave progressively greater hybridization to the heterologous probe and is believed to be due to low levels of expression of such RNA. Such hybridization was specific, since RNA from species not closely related to the rat (e.g., Rhesus monkey) failed to exhibit similar behavior. The WFU-RaLV was spontaneously produced by W/FU rat cells, and the KiMSV (KiMuLV) was grown in NRK.

7 VOL. 17, 1976 z 2 I-- a RE I.- z U Ī A i C,t (mole x sec/liter) FIG. 4. Comparison of rat-specific KiMSV sequences with those of an endogenous RaLV in NRK. Hybridization of NRK cellular RNA was to the [3HJDNA products of KiMSV (KiMuLV) grown in NRK (- -) and of WFU-RaLV (-). Hybridization was as described in the footnote to Table 1. The RNAs used were from NRK (0, U), and from NRK spontaneously producing endogenous RaLV (0, 0). cells were found to not be the characteristic 35S. As illustrated in Fig. 5, 70S viral RNA prepared from NRK-grown KiMuLV was formamide dissociated and sedimented on a sucrose gradient. Hybridization to [3H ]DNA prepared from NIHgrown KiMuLV showed the KiMuLV RNA sequences to be the expected 35S. In contrast, hybridization to an NRK-RNA-specific probe (prepared through preannealing NRK RNA to [3H]DNA prepared from NRK-grown KiMuLV) revealed the rat RNA sequences packaged by the virus to be substantially smaller, with a broad peak in the range of 20-30S. These packaged RNA sequences are presumably the same 30S RNA species detected by Tsuchida et al. (42) within NRK cells. The smaller size is not a peculiar characteristic of rat leukemia viruses, since dissociated 70S RNA prepared from the WFU-RaLV had the expected 35S subunit size (unpublished observation). If a portion of such rescuable rat sequences now represents an integral portion of the KiMSV genome, one would expect KiMSV RNA to show cosedimentation of rat sequences with KiMuLV sequences. To avoid confusion with packaged rat sequences or helper leukemia virus sequences (24) it was necessary to examine KiMSV-infected cell RNA instead of KiMSV virus RNA. We thus analyzed RNA prepared from KiMSV-infected transformed nonproducer cells. When denatured K-NIH cell RNA was sedimented on a sucrose gradient and 0 0 VIRUS RNA IN RAT TUMORS 341 subsequently hybridized to [3H ]DNA specific for KiMuLV and for the KiMSV-rat sequences, both types of sequences were now found to cosediment at 30S (Fig. 6). These experiments provide evidence that rat sequences are physically incorporated into the KiMSV genome. Endogenous type C leukemia and MSVspecific rat cellular sequences in rat tissue DNA. If endogenous leukemia viral genes and/or those cellular sequences incorporated in two rat-derived murine sarcoma genomes play a role in the development of rat malignancies, then one would expect to detect an increased specific gene activity in tumor tissues. Three likely sites for increased gene activity include gene amplification at the DNA level, altered transcription to RNA, and changes at the translational level (47). With the gene products corresponding to those rat sequences in the MSV genomes unknown, we were limited to analyzing tumor and normal tissue DNA and RNA. 40- Z I 010~~~~~~~~0 Cr~ ~ ~II W~~~ 9L20 o. n 28S 18S FRACTION NUMBER FIG. 5. Sedimentation behavior of heat-dissociated 70S KiMuLV-NRK viral RNA. 70S RNA prepared from KiMuL V grown in NRK was isolated by centrifugation for 2 h at 40,000 rpm on a 5 to 20% sucrose gradient in a Beckman SW41 rotor. The 70S RNA was then dissociated by heating 10 min at 37 C in a solution containing 20% formamide (41) before centrifugation for 3 h at 40,000 rpm in a 12.5 to 25% sucrose gradient. Ribosomal RNA markers were centrifuged in parallel. Hybridization to [3H]DNA prepared from KiMuL Vgrown in NIH (0); hybridization to a NRK-RNA-specific (3HIDNA probe, prepared from the DNA product of KiMuLVgrown in NRK by preannealing as described in the footnote to Table 2 (0). i i

8 342 ANDERSON AND ROBBINS z N 020 z w w a. I I 28S 5 10 I5 FRACTION NUMBER FIG. 6. Sedimentation behavior of KiMSV homologous RNA isolated from KiMSV-transformed nonproducer K-NIH cells. Cell RNA was prepared by the method of Tsuchida et al. (43), denatured in formamide, and sedimented in sucrose as described in the legend to Figure. 5. Hybridization to [3HjDNA prepared from NIH-grown KiMuLV (0); hybridization to an NRK-RNA-specific [3H]DNA probe prepared by preannealing of the DNA product of NRK-grown KiMSV (KiMuLV) (-). In hybridization with tissue DNA, sequences homologous to more than 70% of the WFU- RaLV [3H ]DNA probe were seen in all rat strains examined: Osborne-Mendel, Fischer, Wistar/Furth, Copenhagen, Marshall, and Sprague-Dawley (Fig. 7). Similarly, DNA sequences homologous to over 80% of the ratspecific fraction of the KiMSV [3H ]DNA probe were also found in these same rat strains. Both types of sequences are clearly endogenous to the rat. As illustrated for representative samples in Fig. 7A, DNA from rat tumors contains concentrations of WFU-RaLV sequences indistinguishable from the level seen in control tissues, with a Cot½ of 1 x 103. Similarly, the concentration of KiMSV-rat sequences is the same in tumor DNA and control tissue DNA, with a Cot½ of 3 x 102 (Fig. 7B). Total cellular DNA under these hybridization conditions (38% formamide, 43 C, etc.) had a reassociation Cot, of 1 x 104. As molar concentration is inversely proportional to Cot½ (7, 8), and assuming the association of cellular DNA reflects one copy of each gene per haploid control cell, these results suggest there are approximately 10 copies of the WFU-RaLV genome per haploid cell, and 30 of the KiMSV- 4 I l1s J. VIROL. rat sequences. If the WFU-RaLV or KiMSV-rat sequences play a role in tumorigenesis, these results indicate it is not due to gene amplification at the cellular DNA level. RNA of normal rat tissues. To determine if elevated expression of endogenous leukemia virus or MSV-specific rat cellular sequences occurs in rat tumor RNA, it was first necessary to define the basal levels of these sequences in normal rat tissues. Tissue RNAs were thus hybridized to the ['H]DNA product of WFU- RaLV and to the [3H]DNA product of KiMSV (KiMuLV). RNA was prepared from diverse rat tissues of the O-M, Fischer, and W/FU strains, with additional RNAs prepared from Sprague-Dawley, Marshall, and Buffalo rats (Table 4). RNA was isolated from rats of both sexes, ranging in age from 4 weeks to 18 months. No major differences appeared regarding strain, sex, or age (data not shown). RNA was also analyzed from normal tissues of each tumorbearing rat. These control tissues gave results indistinguishable from those obtained from normal animals. Three classes of tissues were distinguishable. A large majority of all tissues examined fell in a first class, having low concentrations of RNA corresponding to both the MSV-rat sequences (Crt20% > 1.5 x 103) and to WFU-RaLV (Ct2o% > 5 x 10'). Tissues of this class fell within the extremes defined by thymus and liver, and representative results are presented in Fig. 8A and B. A second class, reproductive tissues, had elevated concentrations of both types of RNA sequences. In testicle, ovary, and placenta, RNA ultimately hybridizing more than 70% of the WFU-RaLV DNA product was detected at concentrations more than eightfold greater than the first class of tissues. The concentration of the entire group of MSV-rat RNA sequences was elevated to a similar extent (Fig. 8C and D). A third class, consisting of uterus and heart, had high levels of the MSV-rat sequences comparable to the leve' seen in the second class of tissues, but levels of the RaLV sequences were indistinguishable from the low levels seen in the first class of tissues (Fig. 8C and D). Comparison of normal tissues with tumor tissues is complicated by the elevated rates of cell division in the latter. To help ascertain if such growth rates influence the concentration of either the MSV-rat or RaLV RNA sequences, liver regenerating after partial hepatectomy was compared with normal liver. No increase in the concentration of either type of sequences was observed (Table 4). RNA from solid tumors. RNAs isolated from

9 VOL. 17, 1976 VIRUS RNA IN RAT TUMORS 343 Z 0 OU / 9-, 0 Y6-Y I I Cot (mole x sec/liter) FIG. 7. Similar concentrations of MSV-rat sequences and type C rat leukemia sequences in the DNAs of tumor and control rat tissues. DNA/DNA hybridization was at 43 C in a 0.05-ml reaction mixture containing 0.15 M NaCI, 0.01 M Tris (ph 7.5), M EDTA, and 38% formamide. Following incubation for 7 days, nuclease Si was used to measure hybridization. (A) Hybridization of tissue DNA to the [3HJDNA product of WFU-RaLV. Symbols: Fischer rat liver DNA (0), LW-12 leukemic tumor DNA (0), 1082 leukemic liver DNA (A), and Rhesus monkey spleen DNA (y). (B) Hybridization of tissue DNA to the [3HJDNA product of KiMSV (KiMuLV). Results are normalized to the rat-specific fraction of this [3HJDNA probe (40%o of total input). Symbols: Fischer rat liver DNA (-), DMBA-1 mammary adenocarcinoma DNA (A), LW-12 leukemic tumor DNA (U), and Rhesus monkey spleen DNA (V). Hybridization of Fischer rat liver DNA to 'H-labeled NRK rat cellular DNA is included in each figure for comparison purposes (@). the 15 solid-tumor systems listed in Table 5 were hybridized to the [3H]DNA products of KiMSV (KiMuLV) grown in NRK and to the [3H]DNA product of the endogenous rat leukemia virus, WFU-RaLV. For the 10 passaged tumor systems, duplicate tumors were used as RNA sources. As shown in Fig. 9A, seven of the 15 tumors showed MSV-rat RNA levels comparable to the low levels seen in class I normal tissues. Less than 50% hybridization was seen at Crt values up to 104. Similarly, the concentration of endogenous RaLV RNA sequences in these seven tumors was indistinguishable from the low level shown by class I normal tissues (Fig. 9B). Here, less than 20% hybridization was seen at Crt values up to 10'. In a second group of six solid tumors, there was a 5- to 100-fold elevation in the concentration of the MSV-rat RNA sequences, as compared to class I normal tissues, whereas the RaLV RNA concentrations appeared to range from normal to up to 10-fold elevated over class I normal tissues (Fig. 9C and D). Two of these tumors, the DMBA-1 mammary adenocarcinoma and the WR-123 interstitial cell tumor, showed a particular similarity to the class III normal tissues, expressing high levels of the MSV-rat sequences and low levels of RaLV sequences. An additional two of the 15 solid tumors, the Novikoff hepatoma (ascites) and AA-ascites, showed concentrations of both MSV-rat RNA sequences and of RaLV RNA sequences elevated approximately 1,000-fold over the basal level seen in class I normal tissues (Fig. 10). The Ct% for both types of sequences was approximately 102 in each of these ascites tumor RNAs. These unusually high virus-specific RNA levels may reflect type C virus production, since this has been seen by electron microscopic examination of the Novikoff hepatoma (20), and both ascites fluids gave a positive reverse transcriptase assay for type C virus production (data not -hown). Hematologic tumors. RNA was isolated from the five rat leukemias and two rat lymphomas described in Table 5, none of which gave evidence of type C virus production (data not shown). This RNA was again assayed for the presence of MSV-rat sequences and type C

10 344 ANDERSON AND ROBBINS TABLE 4. Endogenous type C RaL V and MSV-specific rat cellular sequences expressed in the RNA of normal rat tissues Class Tissue Rat strain" I. RaLV (_) Thymus F, O-M MSV-rat ( ) Spleen F, O-M, W/FU, M, B, S-D, C Liver F, O-M, W/FU, M, B, S-D, C Regenerating liver F, W/FU Kidney F, O-M Lung O-M, W/FU Brain O-M Leg muscle F, O-M Fetus O-M II. RaLV (+) Placenta O-M MSV-rat (±) Ovary F, O-M Testes F III. RaLV ( ) Uterus (virgin) F, O-M MSV-rat (+) Heart F a Rat tissue was hybridized to the reverse transcription [sh JDNA products of WFU-RaLV and KiMSV (KiMuLV), as described in text. bduplicates of each tissue were analyzed. F, F-344 Fischer; S-D, Sprague-Dawley; C, Copenhagen; M, Marshall; B, Buffalo; O-M, Osborne-Mendel; W/FIT, Wistar Furth. cralv (- ) is defined as less than 20% hybridization by tissue RNA of the ['H]DNA product of WFU-RaLV at Crt values up to 10'. MSV-rat ( -) is defined as where cell RNA hybridized less than 20% of the rat-specific fraction of the KiMSV (KiMuLV) ['HI]DNA, or less than 8% of the total ['HIDNA, at a Crt up to 10'. Representative hybridization results are presented graphically in Fig. 3. J. VIROL. leukemia viral sequences by hybridization to the [3H ]DNA products of KiMSV (KiMuLV) and of WFU-RaLV. In four of the five leukemias and in one of the two lymphomas, the concentration of MSV-rat RNA sequences was more than 10-fold greater than that of class I normal tissues (Fig. lla and C). Hybridization to the RaLV probe revealed that in the same four of the five leukemias and in the same lymphoma, there was a comparable elevation of concentration of some leukemia viral sequences (Fig. l1b and D). However, there was clear evidence of saturation being achieved at only 30 to 60% hybridization of the WFU-RaLV [3H ]DNA. These plateaus were not artifacts of hybridization, since low levels of WFU-RaLV viral RNA added to the leukemic RNAs sufficed to create total hybridization, and low-level RaLV producers (with Crt½ values of 5 x 103) showed complete hybridization of the same probe (data not shown). To determine if common RaLV sequences were being expressed by each leukemia, additivity hybridizations were performed (Table 6). No additivity was seen; the lesser member of any pair was found incapable of showing any increase beyond that given by the greater member in hybridization with the WFU-RaLV [3H ]DNA. One possible explanation for this result could be that the RaLV RNA sequences detected share one common end and terminate prematurely on the endogenous viral genome, at points particular to each tumor. DISCUSSION These studies were undertaken to further define the nature of those rat sequences present in the KiMSV and HaMSV genomes and to determine if these rat sequences may play a significant role in spontaneous rat tumors. Type C viruses are known to package diverse RNA species. Specific host trna and rrna has been found (40), as well as low levels of globin mrna associated with 70S RNA (17). In these cases, such RNA is not covalently associated with viral RNA. Low levels of endogenous viral RNA have also been shown to be picked up by growth of exogenous virus in cat cells (6). Type C leukemia viruses also can package the RNA of, and provide helper functions necessary for, the replication of murine sarcoma viruses. Type C viral reverse transcriptases have been shown to accept readily nonviral RNA as a template for DNA synthesis (30). In line with these findings, we have shown that growth of diverse type C viruses in rat cells can result in substantial packaging of host RNA. Rat-specific sequences can constitute over 70% of the DNA product synthesized by rat-grown viruses. This finding alone makes it clear that extreme care must be taken in evaluating genetic relatedness studies of type C viruses, particularly when grown in rat cells. The KiMSV and HaMSV genomes both contain approximately half of those packaged host sequences found in the DNA products of type C leukemia viruses grown in rat cells. This observation has also been reported in the case of Moloney MuLV grown in NRK (34). Our results further indicate that these rat sequences are now covalently attached to the sarcoma virus genomes, since they cosediment under denaturing conditions and propagate with the sarcoma viruses. A schematic presentation of how the packaging of cell sequences relates to the genomes of the murine sarcoma viruses is presented in Fig. 12. Homology seen between KiMSV RNA or HaMSV RNA and the DNA product of a RaLV grown in NRK has led to the repeated assumption that some or all of the rat sequences in these sarcoma virus genomes represent type C rat leukemia virus sequences (24, 35, 37, 42). However, as has been stated by Scolnick et al.

11 VOL. 17, 1976 VIRUS RNA IN RAT TUMORS I I IT I I (A) - (B) FSo N aco x I- z U I- ILl 40F (C) ~ 01 L/ 1' 0,al- 0---" i- i l i I-, / " / I/ /P/i./ / la~*.//,, / //, (D) IV. //,; ' O i Crt (mole x sec/liter) FIG. 8. Hybridization of RNA prepared from normal rat tissues to the [3H]DNA products of KiMSV (KiMuLV) and of WFU-RaLV. DNA-RNA hybridization conditions were as described in the text with 2,000 counts/min of KiMSV (KiMuLV) [3H]DNA or 1,000 counts/min of WFU-RaLV [3HJDNA being annealed to the tissue RNAs. Hybridization to KiMSV (KiMuLV) ['3HJDNA is normalized to the rat-specific fraction of this probe (40% of input) as determined by hybridization to NRK RNA. Hybridization to the WFU-RaLV [3HIDNA is presented directly. Hybridization of RNA from representative class I normal rat tissues (A) to KiMSV (KiMuLV) [3HJDNA, and (B) to WFU-RaLV [3HJDNA. Symbols: Fischer thymus (V), MTW9a nontumored spleen (0), O-M kidney (0), and O-M liver (a). Composites of the results obtained from all class I tissue RNAs for each probe are indicated by (... ). Hybridization of RNA from representative class II and class III normal rtdt tissues (C) to KiMSV (KiMuLV) [3H]DNA and (D) to WFU-RaLV [3HJDNA. Symbols: class I: O-M placenta (0), Fischer testes (U); class III: O-M uterus (virgin) (y), Fischer heart (a). The composites of class I tissue hybridization results obtained with each DNA probe are included for purposes of comparison (...). 1)I.1 (34), this is far from a rigorous proof; by the same reasoning, globin genes could be taken to be type C viral. It should now be readily apparent that the DNA product of most type C viruses grown in NRK, not just NRK-RaLV, will show homology with these sarcoma viruses. RaLV produced by cells other than NRK failed to show homology with the sarcoma viruses. In the case of the NRK-RaLV, spontaneous virus production massively increases the concentration of RNA homologous to other known rat leukemias, but has no effect on the MSV homologous RNA concentration. This finding, consistent with the idea that the NRK- RaLV also is totally unrelated to the MSV sequences, has also been reported in another

12 346 ANDERSON AND ROBBINS TABLE 5. Rat tumor systems examined for expression of type C RaLV- and MSV-specific rat cellular sequences0 Tumor Expressed as Trans- Primary derived RNA" Tumors Designation Strain planted tumor cell M tumor culture RaLV MV rat Solid tumors Carcinomas Mammary adenocarcinomas MT/W9a W/FU x + + MT/W449a W/FU x + R-3230 Fischer x DMBA-1 Fischer x + V R-157 Lewis x + + Prostate adenocarcinoma R-3327 Copenhagen 2331 x Hepatoma Novikoff (as- Wistar x cites) Spontaneous ascites AA Wistar x Chemically induced carci- DMBA-A Sprague-Dawley x nomas DMBA-B Sprague-Dawley x Epidermoid carcinoma WR-122 ACI x + + Sarcomas Fibrosarcoma CSE Fischer x Rhabdomyosarcoma NS104 Fischer x Other Glial tumor GBTW Wistar x Interstitial cell tumor WR-123 Fischer x _ + + Hematologic tumors Leukemias Acute myelogenous Dunning Fischer x + + Chronic myelogenous LW-12 W/FU x + + Acute monocytic R-3149 Fischer x + + Lymphocytic WR-6 W/FU x Mononuclear cell (18) 1082-F Fischer x + + Lymphomas Lymphoma L-8 W/FU x Lymphosarcoma Murphy-Sturm Wistar j x + + a DNA and RNA were isolated from each tumor system. These nucleic acids were then hybridized to the [3H 1DNA product of the WFU-RaLV and of KiMSV (KiMuLV), as described in the text. DNA from all tumor systems showed no variation in the concentrations of either type of sequences (see text). b In hybridizations of the tissue RNAs, + denotes more than 20% hybridization was achieved at a Crt value of 10,, and + + indicates more than 50% hybridization was achieved at a Ct of 2 x 102. Actual hybridization results are presented in Fig. 9, 10, and 11. J. VIROL. spontaneously virus-producing NRK clone (34). We further found the MSV-homologous rat RNA, as packaged in type C virions grown in NRK, is only 20-30S, whereas 35S viral RNA is characteristic of endogenous mammalian type C viruses (40). This finding, which could also be explained by a deletion mutation of a rat type C virus, is consistent with the report by Tsuchida et al. (42), who found that intracellularly in NRK a 30S RNA species is seen that is homologous to KiMSV. All together, these results support the hypothesis that those rat sequences in the KiMSV and HaMSV genomes are not those of an endogenous rat type C leukemia virus, but instead are other cellular sequences. Until the precise role of these rat sequences is defined, it will be possible to still argue that the rat sequences of KiMSV and HaMSV could represent those of a rat type C virus. However, such a putative virus would have to be genetically unrelated to all other presently available rat type C viruses, would have a genome significantly smaller than other endogenous type C leukemia viruses, and would have to be defective in that no virus is produced although such RNA is present in some cells at concentrations corresponding to high levels of normal type C virus production (manuscript in preparation). Our analyses of rat tumors and control rat tissues show that within rat cellular DNA reside both type C leukemia viral genetic information and those rat cellular sequences that were incorporated in the genomes of the KiMSV and HaMSV. Though we found no evidence of variation at the DNA level for either type of sequences, both in diverse normal tissues and in tumors, the potential for involvement of such endogenous genes in tumorigenesis clearly exists. Expression of the endogenous rat leukemia

13 VOL. 17, 1976 viral and of the MSV-rat sequences showed substantial variation at the cell RNA level. Three patterns were observed in normal rat tissues alone. Most tissue RNA contained barely detectable amounts of either leukemia viral RNA or the MSV-rat RNA sequences. Complete hybridization of the [3H ]DNA product of WFU-RaLV or of the rat-specific fraction of KiMSV (KiMuLV) was not achieved, even at Cot values exceeding 5 x 104. In contrast, reproductive tissue RNA showed over eightfold elevation of the concentration of both types of RNA sequences, with nearly complete hybridization of each DNA probe being achieved at Crt values of 2 x 104. Two tissues (heart and uterus) showed 10-fold elevated concentrations of the (A) 80 Go C z 0 o _ 2 0 r 0~~~~ IL C.) X L 0 X (C) _ VIRUS RNA IN RAT TUMORS 347 MSV-rat sequences alone. This detection of endogenous leukemia viral RNA at elevated concentrations in some normal tissues indicates that lessened control of these viruses does not invariably lead to tumorigenesis, an observation consistent with the detection in mice and baboons of complete type C virus in normal embryo tissue (19, 46). By the same reasoning, if there is an oncogenic activity in the MSV-rat sequences, such activity must require more than just elevated RNA levels. This argument also has been used in regard to the NRK cell line, which is not transformed but which does contain high concentrations of MSV-rat sequences (34, 42). Seven of 15 solid tumors and two of seven C t (mole x sec/liter) FIG. 9. Hybridization of RNA prepared from rat sarcomas, carcinomas, and other solid tumors to the [3H]DNA products of KiMSV (KiMuLV) and of WFU-RaLV. Descriptions of each tumor are given in Table 5. Conditions were as described in the text and in the legend to Fig. 8. The curves representing the average of the results obtained with class I normal tissue RNAs are included for purposes of comparison ( --.). RNA from rat tumors with low concentrations of both types of rat sequences, hybridized to the [3HJDNA product of (A) KiMSV (KiMuLV), and (B) WFU (RaLV). Symbols: GBTW (a), NslO4 (0), CSE (U), R-3327 ((D),'R-3230 (0), DMBA-A (0), and DMBA-B (A). RNA from rat tumors showing higher concentrations of either type of rat sequences, hybridized (C) to KiMSV (KiMuLV) [3H]DNA and (D) to WFU-RaLV [3H]DNA. Symbols: MT/W9a (-), DMBA-1- (U), WR-123 (0), WR-122 (a), WR-157 (A), and MT/W 449a (V).

14 348 ANDERSON AND ROBBINS J. VIROL. z 0 N a I.- z LU IL' Ct (mole x sec/liter) FIG. 10. Hybridization of rat ascites tumor RNAs to the [8HJDNA products of (A) KiMSV (KiMuLV) and (B) WFU-RaL V. Conditions were as described in the text and legend to Fig. 8. For comparison purposes, the composite curves obtained with class I normal tissue RNAs are included (...). Symbols: AA ascites (U), Novikoff hepatoma ascites (0). hematologic tumors showed no evidence of increased expression at the RNA level either of endogenous leukemia virus or of MSV-rat cellular sequences. By this criterion, we thus obtained no evidence for involvement of either type of sequences in almost half of the tumors we examined. In striking contrast, two ascites tumors showed both types of RNA at concentrations more than 100-fold greater than that seen in most normal tissues. A remaining group of six solid tumors and five hematologic tumors showed intermediate increases in both types of sequences. The type C sarcoma viruses offer a useful model for the speculative interpretation of our data. Both the Kirsten and Harvey rat-derived sarcoma viruses, along with the Moloney mouse-derived sarcoma virus, only contain around half of their parental leukemia virus genomes (33). In a series of leukemic rat tissues where cell DNA hybridized more than 70% of our RaLV [3H]DNA probe, RNA from these same tissues showed elevated concentrations of RaLV sequences hybridizing at saturation only 30 to 60% of the same DNA probe. This would be consistent with only partial expression of a specific endogenous RaLV. In these leukemic rat tissues, we also found elevated expression of those same rat sequences present in the ratderived murine sarcoma virus genomes. One possible explanation of these findings is that recombination of the two types of sequences has occurred, creating a transforming principle analogous to the murine sarcoma viruses. Similarly, in the two ascites tumors we found massive expression of RNA homologous to the entire RaLV probe along with the MSV-rat sequences. With complete type C virus production also indicated, here it is possible that recombination has created a competent sarcoma virus analogous to the avian sarcoma viruses (11, 29). These interpretations are currently under further study. The elevated MSV-rat and/or RaLV concentrations seen in these tumors and in certain normal tissues could reflect an absolute increase in the amounts of each RNA, a decrease in the amounts of other cellular RNA species, or a combination of both of these factors. The fact that many tumors and some control tissues had elevated levels of one but not both species of RNA would indicate that the specific RNA increases seen were absolute. As discussed above, gene amplification at the DNA level cannot be responsible for those elevated RNA levels detected. This leaves us with two possibilities: either specific transcription rates are increased or else some form of RNA stabilization is occurring. It is impossible for analysis of tumor and normal tissue nucleic acid alone to prove or disprove the oncogenic nature of any given set of genes. In view of the presence of two genetically unrelated endogenous leukemia viruses in the cat (6), negative results could be due to the failure of the [3H]DNA probes used in these studies to detect expression of unknown but analogous endogenous viral- or MSV-rat-like sequences. It is also conceivable that the critical controlling point in oncogenic activity is post-

15 VOL. 17, sof 60F VIRUS RNA IN RAT TUMORS 349 (A) ( I I I (A) (B) z 0F N a I.- z us a: 40 20F o 60~ 40k 20~ I4fi Crt (mole x sec/liter) FIG. 11. Hybridization of RNA prepared from rat leukemias and lymphomas to the ['H]DNA product (A, C) of KiMSV (KiMuLV) and (B, D) of WFU-RaLV. Conditions were as described in the text and legend to Fig. 8. The composite curves obtained with class I normal tissues RNAs are included for comparison (... ). Symbols for leukemic tumor RNAs: LW-12 (0), Dunning (0), R-3149 (0), 1082-F (A), and WR-6 (0); for lymphoma tumor RNAs: L-8 (0), and Murphy-Sturm (v). TABLE 6. Lack of additive effects of leukemic tumor RNA in hybridization to WFU-RaLV [3HJDNAa Hybridization (%) to WFU-RaLV [3H]DNA RNA sources 1082 WFU Fischer R3149 Dunning LW12 producer 1082 Fischer NTb NT R NT Dunning NT LW WFU-RaLV 98 producer '5- (. I (C) asaturating levels of leukemic tumor or RaLV-producing cell RNA (150 to 500 gg), alone and in the indicated combinations, were hybridized to the [3H]DNA product of WFU-RaLV. Conditions of hybridization and assay were as described in the text. b NT, Not tested. // (D), He * w~~7s,-/y 4 -- transcriptional. Assuming our probes do detect all relevant sequences, these studies cannot rule out other conceivable alterations in the expression of either type of sequences. The use of viral [3'H]DNA products prepared in the presence of actinomycin D precludes detection of RNA expressed as the (-) strand. The inability of many control tissue RNAs to hybridize more than 50% of either probe at the maximum Crt values tested (though no evidence of saturation was seen in these cases) leaves open the possibility that in many tissues only limited portions of either type of sequences may be transcribed, and that in tumors a larger percentage is transcribed. Lastly, and perhaps most seriously, it is known that transformed cells transcribe a proportion of their DNA greater than that seen in normal cells (12). In the case of the mouse, - 5

16 350 ANDERSON AND ROBBINS TYPE-C MuLV d b c d e f CELLULAR SEQLUENCES V W X y Z I..4 R' / R -J J. VIROL. ~~~~~~~~~~~~~~~~~~~~~~~~~I SARCOMA VIRUS d b C y z 1.1 M - 4A R - l FIG. 12. The apparent relationship of packaged rat sequences to those rat sequences in the KiMSVgenome. Type C KiMuLV growing in rat cells readily packages a discrete segment of rat RNA, RR'. Through a recombinational event at an unknown level, a genetic hybrid (KiMSV) was created that contains some type C leukemia viral genes of mouse origin (M) and some cellular genes of rat origin (R). this was found to be at least 15% of the cell DNA transcribed in transformed cells, whereas only 8.5% was seen in normal cells. Thus it is clearly possible that the elevated concentrations of endogenous leukemia viral or MSV-rat cellular RNA sequences could be a secondary effect of transformation. In spite of the above limitations, it is clear that transforming activity somehow resides in sarcoma virus genomes composed of leukemia viral and specific cellular sequences. Our results provide evidence supporting the hypothesis that processes analogous to those that generated the transforming activity associated with type C murine sarcoma viruses may also be responsible for many (but not all) spontaneous and carcinogen-induced rat malignancies. Studies are currently in progress to clarify the relationship between the MSV-rat sequences and the RaLV sequences, as they are expressed in tumor RNAs, and to further define the normal functions of both types of sequences. ACKNOWLEDGMENTS We wish to thank William Hagen, Mary Cothran, Claude Davis, and Claire Dunn for their technical assistance. This work was performed in the laboratory of Stuart Aaronson. We wish to acknowledge valuable conversations with George Todaro, Cy Cabradilla, and Maya Piniero. Paula Pluka at the Mason Research Institute was most helpful in providing tumors. This work was supported by a contract from the Virus Cancer Program of the National Cancer Institute. G. Anderson was supported in part by postdoctoral fellowship PF-721 from the American Cancer Society. ADDENDUM Tsuchida et al. (43) have recently reported an analysis of RNA species in a hamster tumor line that was induced by Harvey sarcoma virus, and now is also producing a type C hamster virus. These studies have also indicated that the Harvey sarcoma virus contains a single 30S RNA species composed of mouse leukemia virus sequences recombined with rat sequences. LITERATURE CITED 1. Aaronson, S. A Isolation of a rat-tropic helper virus from M-MSV-O stocks. Virology 44: Aaronson, S. A., and W. P. Rowe Nonproducer clones of murine sarcoma virus transformed BALB/3T3 cells. Virology 42: Aaronson, S. A., and C. Weaver Characterization of murine sarcoma virus (Kirsten) by transformation of mouse and human cells. J. Gen. Virol. 13: Benveniste, R. E., R. Heinemann, G. L. Wilson, R. Callahan, and G. J. Todaro Detection of baboon type-c viral sequences in various primate tissues by molecular hybridization. J. Virol. 14: Benveniste, R. E., and E. M. Scolnick RNA in mammalian sarcoma virus transformed nonproducer cells homologous to murine leukemia virus RNA. Virology 51: Benveniste, R. E., and G. J. Todaro Homology between type-c viruses of various species as determined by molecular hybridization. Proc. Nat]. Acad. Sci. U.S.A. 70: Birnstiel, M. L., B. H. Sells, and I. F. Purdom Kinetic complexity of RNA molecules. J. Mol. Biol. 63: Bishop, J The effect of genetic complexity on the time course of RNA-DNA hybridization. Biochem. J. 113: Chattopadhyay, S. K., D. R. Lowy, N. M. Teich, A. S. Levine, and W. P. Rowe Evidence that the AKR murine leukemia virus genome is complete in the DNA of the high-virus AKR mouse and incomplete in the DNA of the virus negative NIH mouse. Proc. Nati. Acad. Sci. U.S.A. 71: Duc-Nguyen, J., E. M. Rosenblum, and R. F. Zeigel Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92: Duesberg, P., and P. Vogt Differences between the ribonucleic acids of transforming and nontransforming avian tumor viruses. Proc. Natl. Acad. Sci. U'.S.A. 67: Grady, L. J., and W. P. Campbell Non-repetitive DNA transcription in mouse cells grown in tissue culture. Nature (London) New Biol. 243:

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