Vol. 66, No. 7

JOURNAL OF VIROLOGY, JUlY 1992, p. 4377-4381

0022-538X/92/074377-05$02.00/o

Copyright © 1992, American Society for Microbiology

Enhanced Gene Expression of the Murine Ecotropic Retroviral Receptor and Its Human Homolog in Proliferating Cells TAKAYUKI YOSHIMOTO,t ERIKO YOSHIMOTO, AND DANIEL MERUELO*

Department of Pathology, New York University Medical Center, 550 First Avenue, New York, New York 10016 Received 4 February 1992/Accepted 15 April 1992

The receptor for gp7O envelope glycoprotein of murine ecotropic leukemia virus is essential for virus entry into the host cell and has been recently demonstrated to function as a cationic amino acid transporter. In the experiments reported herein, we compared the gene expression of the murine ecotropic retroviral receptor (ERR) and its human homolog (H13) in rapidly proliferating cells versus resting cells using four different systems. (i) The expression of ERR gene is enhanced during activation of T and B lymphocytes by concanavalin A and lipopolysaccharide, respectively. Similar enhancement is observed by adding phorbol 12-myristate 13-acetate (PMA) or calcium ionophore (A23187). These phenomena appear to involve protein kinase C; two PMA analogs, 4a-phorbol and 4ca-PMA, lacking the ability to activate protein kinase C fail to induce elevated levels of gene expression, and the protein kinase C inhibitor, H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloridel, inhibits the enhancement induced by PMA. (ii) Friend murine leukemia virus induces rapid splenomegaly and acute erythroleukemia in sensitive mice. Concomitantly with splenomegaly, ERR gene expression in spleen cells increases dramatically. (iii) The level of expression of the ERR or H13 gene in a variety of tumor cells is highly elevated compared with the level in noncancerous cells. (iv) H13 gene expression decreases upon terminal differentiation of the human promyelocytic leukemia cell line HL-60 into granulocytes or macrophages by dimethyl sulfoxide or PMA, respectively. These results suggest that ERR and H13 genes play an important role in cellular proliferation. refractory to retroviral infection (10, 29, 31). These phenomhave been thought to be due to the absence of a receptor for ecotropic retroviruses in adult quiescent hepatocytes and the presence of the receptor in fetal and regenerating hepatocytes, which are rapidly proliferating. These facts led us to investigate the gene expression of ERR and H13. We first observed that ERR gene expression was markedly enhanced in T and B lymphocytes activated by stimulation with ConA and lipopolysaccharide (LPS), respectively. Expression of ERR and H13 was also examined in vivo in rapidly proliferating cells infected by Friend leukemia virus (FV) in a variety of other tumor cells versus their noncancerous cell counterparts and in the differentiating human promyelocytic leukemia cell line HL-60. In each case, the level of expression of the ERR or H13 gene is highly elevated in rapidly proliferating cells, suggesting an important role for ERR and H13 gene expression in cellular proliferation.

More than 70 genes or gene products are known to increase in expression when T cells become activated in response to antigen and self-histocompatibility molecules on the surfaces of antigen-presenting cells (6). Polyclonal activators such as lectins, calcium ionophores, or antibodies to the T-cell receptor for antigen can mimic the response induced by antigen. Some of the genes whose expression increase encode cytokines and their receptors, some encode nuclear regulatory proteins, and still others are involved in the transport of ions and nutrients into cells to prepare them for growth. Some proto-oncogenes, which have regulatory roles in normal cellular proliferation and differentiation (7, 12, 16, 24, 27), also have elevated levels of expression during T-cell activation. In addition, they may later contribute to neoplastic transformation. Murine ecotropic retroviral receptor (ERR) was molecularly cloned by Albritton et al. (1) and has been demonstrated to function as a cationic amino acid transporter (17, 28). Recently, we cloned a human gene (H13) homologous to the murine ERR gene, with 87.6% identity at the amino acid level (32). The H13 gene is also fairly homologous to the murine T-cell early activation gene (TEA) with 52.5% identity at the amino acid level. TEA is expressed by concanavalin A (ConA)-activated lymphocytes, but its function remains unknown (18). TEA is the first example of a gene encoding a multiple transmembrane-spanning protein which is induced during T-cell activation. It has been known for several years that hepatocytes can be infected by ecotropic retroviruses during fetal development and liver regeneration, although adult hepatocytes are

ena

MATERIALS AND METHODS

Mice. All mice used in this study were bred in

our

colony

at the New York University Medical Center (New York,

N.Y.). Reagents. Phorbol 12-myristate 13-acetate (PMA), 4orPMA and 4oa-phorbol were obtained from GIBCO BRL (Gaithersburg, Md.). H7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride], A23187, and LPS (Escherichia coli 055:B5) were obtained from Sigma (St. Louis, Mo.). Cells. In vitro-adapted tumors (radiation leukemia virusinduced tumors and X-irradiation-induced tumors) of various inbred mouse strains were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) (2, 19, 20). Human acute lymphoblastic leukemia cell line CCRF-CEM (ATCC CCL119) and promyelocytic leukemia cell line HL-60 (ATCC CCL240) were

Corresponding author. t Present address: Department of Allergology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minatoku, Tokyo 108, Japan. *

4377

4378

YOSHIMOTO ET AL.

(A)

(on A 1I 6 12 24 hr

+

J. VIROL.

(B) + (.) II 6

(C)

12

l2 4 r

41

3

O 6

12 h}r

.;..-..... i "am

12 hlr

0

3

12 hr

6

.11 il

.

5.O.

D. O -

2.0B-,kctin-

2.0

maintained in RPMI 1640 medium supplemented with 10 and 20% FBS, respectively. Human kidney carcinoma cell line A-498 (ATCC HTB44), lung carcinoma cell line A-427 (ATCC HTB53), and colon adenocarcinoma cell line CaCo-2 (ATCC HTB37) were maintained in minimum essential medium supplemented with 10, 20, and 20% FBS, respectively. Splenocytes or thymocytes obtained from BALB/c mice at 4 to 10 weeks of age were seeded at 5 x 106 to 8 x 106 cells per ml in RPMI 1640 medium supplemented with 10% FBS and 0.05 mM ,B-mercaptoethanol, and then the cells were stimulated with 10 ,g of ConA per ml, 50 ,ug of LPS per ml, 50 ng of PMA per ml, or 0.5 ,uM A23187. HL-60 cells were stimulated with 50 ng of PMA per ml or 1.25% (vol/vol) dimethyl sulfoxide. Probes. The DNA probes used in this study were as follows: ERR cDNA probe of BamHI-EcoRI fragment of pJET (1) which was kindly provided by L. M. Albritton and J. M. Cunningham (Harvard Medical School, Boston, Mass.), H13 cDNA probe of NruI-PstI fragment (32), and mouse and human 1-actin probes (5). Proliferation assay. Cultures were pulsed with 1 ,uCi of [3H]thymidine (New England Nuclear, Boston, Mass.) per well for 6 h. The samples were collected with a semiautomatic cell harvester, and the counts per minute were recorded by scintillation counting. FV infection in vivo. FV (8) was initially obtained from F. Lilly (Albert Einstein College of Medicine) (21, 22). The virus was maintained through in vivo passage by homogenizing the enlarged spleen of mouse 10 days after it had been injected with virus intravenously. After 106 focus-forming units of FV was administered intravenously into BALB/c mice, two mice each were sacrificed on days 0, 1, 4, 6, and 11. The spleens were then weighed, and their RNAs were prepared for Northern (RNA) blot analysis by using ERR cDNA as a probe. Northern blot analysis. Total cellular RNA was isolated by the guanidinium isothiocyanate method (3) from cell lines, mouse tissues, or human noncancerous tissues. The RNA samples were electrophoresed through a 1% agarose gel containing formaldehyde, transferred to Zeta-Probe membrane (Bio-Rad, Richmond, Calif.) and subjected to hybridization as described previously (4).

6

,.I.W.11.1

sow_

FIG. 1. Enhancement of ERR gene expression during T- and B-lymphocyte activation. Total cellular RNA was prepared from quiescent and activated splenocytes (A and C) and thymocytes (B) harvested at the indicated times following activation with the T- and B-cell mitogens, ConA (10 p.g/ml) and LPS (50 p.g/ml). After electrophoresis and Northern transfer, ERR transcripts (approximately 9 and 8 kb) were detected by hybridization to 32P-labelled ERR cDNA (BamHI-EcoRI) probe. The lower panel shows the same blot stripped and subsequently probed with murine j3-actin to assess the amount of RNA loaded in each lane. Molecular size markers used are 28S and 18S (5.0 and 2.0 kb) endogenous rRNAs.

3

kb)

k1)

U* rn0

(B))PMNMA

(A)A23187

+ XLPS

A

(

t

FIG. 2. Enhancement of ERR gene expression by A23187 and PMA. Total RNA was prepared from quiescent and activated splenocytes by using the calcium ionophore A23187 (0.5 ,uM) and the tumor-promoting phorbol PMA (50 ng/ml) and subjected to Northern blot hybridization probed with ERR cDNA.

RESULTS

Enhancement of ERR gene expression during T- and B-lymphocyte activation. To investigate ERR gene expression during T-cell activation, ConA was added to splenocytes and thymocytes, and total cellular RNA was prepared after 0, 6, 12, and 24 h of incubation. Figure 1A and B show the hybridization patterns using RNA probed with ERR cDNA. The ERR transcripts (approximately 9 and 8 kb) are barely detectable in quiescent splenocytes, but both of them become strongly expressed, peak at about 6 hr with an approximately 10-fold increase, and then decrease by about 24 h. In contrast, the ERR transcripts in thymocytes slowly increase and reach approximately 10-fold-greater levels at 24 h than at time 0. The enhancement of ERR transcripts was also observed during B-cell activation by LPS, as shown in Fig. 1C. The transcripts rapidly increase and reach their maximum level, with an increase of approximately 10-fold at about 3 h. Involvement of PKC in ERR gene enhancement. Similar enhancement of ERR expression was seen by adding the calcium ionophore, A23187, or PMA to splenocytes (Fig. 2A and B, respectively). A23187 activates protein kinase C (PKC) through elevation of cellular calcium ion concentration, and PMA directly activates PKC by interacting with the

C(otrolL .4~

4u.Phorbol

-~~~~~~~~~11 PX6 O 2 8

(OltlWCI)

2-)

]Th,*iniidine

@t6)-4 4 0 FIG. PMA

the

3.

Involvement of PKC in ERR gene enhancement. Two

analogs, 4,-phorbol (50 nglml) and 4o-PMA (50 nglml), lacking

ability

for 3

Incorp)oraItionl

h.

to activate PKC were added to

In

addition, splenocytes

inhibitor H7 (100

after PMA

were

splenocytes and pretreated with

incubated the

PKC

±M) for 1 h and subsequently incubated for 3 h

(50 ng/ml)

was

splenocytes and subjected

to

was

Northern blot

prepared from analysis, using

these ERR

splenocytes were further incubated for an additional 24 h, and cellular proliferation was determined by [3 measuring H]thymidine incorporation after being pulsed with 1 p.Ci [3 H]thymidine for 6 h (B).

cDNA

as a

probe (A).

added. RNA

These

VOL. 66, 1992

ENHANCED GENE EXPRESSION OF ERR AND H13

(Al)

B11) 1)

I

6

4

1

I1

l'.%

-.

khi 5.0-

0.4

2.0-

(0

1

B-Actin-

4

6

11

Daviater FV I~noculationi expression in FV-

FIG. 4. Incremental increase of ERR gene infected spleen with splenomegaly in vivo. After FV (106 ffu) was administered intravenously into BALB/c mice, two mice were sacrificed on each day indicated and their spleens were weighed (B). Subsequently, RNA from each spleen was prepared and subjected to Northern blot analysis using ERR cDNA as the probe (A).

catalytic site of the enzyme (14). In splenocytes stimulated with A23187, the ERR transcripts slowly increase and reach approximately 10-fold-greater levels at 24 h than at time 0. In contrast, the ERR transcripts rapidly increase and peak at about 3 h, with an approximately 10-fold increase in splenocytes stimulated with PMA. These results indicate the involvement of PKC in ERR gene enhancement. To confirm these observations, we used two PMA analogs, 4a-phorbol and 4a-PMA, which are unable to activate PKC, and the PKC inhibitor, H7, which can inhibit T- and B-cell activation (13, 26). Splenocytes were either incubated with PMA analogs for 3 h or pretreated with H7 for 1 h and subsequently incubated for 3 h with PMA. Figure 3A shows the hybridization pattern of the ERR cDNA probe and RNA prepared from these splenocytes; the results of [3H]thymidine incorporation (Fig. 3B) are also shown. Neither enhancement of ERR gene expression nor stimulation of proliferation was observed with the PMA analogs. Furthermore, H7 inhibited the effects of PMA. These results suggest PKC involvement in the observed increases in ERR gene expression.

Incremental increase of ERR gene expression in FV-infected

(A.\) MIouse

cells in vivo. FV (8) is an aggressive polytropic (mink cell focus-forming virus) retrovirus and induces rapid splenomegaly and acute erythroleukemia in sensitive mice such as the BALB/c strain. The effect of FV-induced splenomegaly on ERR gene expression was examined in vivo. After FV was administered intravenously, two mice each were sacrificed on days 0, 1, 4, 6, and 11. The spleens were weighed, and RNAs were prepared for Northern analysis, using ERR cDNA as a probe. These results are shown in Fig. 4. Spleen weights slowly increase after FV administration and are approximately 10 times higher than those of healthy mice by day 11. This progressing splenomegaly is accompanied by gradual incremental increase in ERR gene expression, reaching >10-fold-higher level by day 11. These results offer evidence that ERR gene expression plays a role in cellular proliferation. Elevated gene expression of ERR or H13 in tumor cells compared with that in noncancerous cells. We next compared ERR or H13 gene expression in a variety of noncancerous and tumor cells. Fractionated X-irradiation- and radiation leukemia virus-induced tumors, as well as a variety of human tumors including an acute lymphoblastic leukemia, a lung carcinoma, a kidney carcinoma, and a colon adenocarcinoma were used. Figure 5 shows results of Northern analyses using RNA prepared from these tumor and noncancerous cells probed with ERR or H13 cDNA. The levels of ERR and H13 gene expression in mouse and human tumor cells are highly elevated compared with those in noncancerous cells, indicating the importance of these genes for cellular proliferation. Decreased expression of H13 gene upon terminal differentiation of HL-60 cells into granulocytes and macrophages. Extensive phylogenetic conservation and differential expression during mouse pre- and postnatal development support the widely held notion that proto-oncogenes participate in normal cellular metabolic processes. Several investigators have demonstrated that the expression of proto-oncogenes is modulated by a variety of agents capable of inducing proliferation and differentiation (23, 30). We examined H13 gene expression following terminal differentiation of HL-60 cells ( B) Htiiiiat

_

X: '4 =-

_

-

kb

kb_

m

Rm

4379

4

m

5;.0-

.0

2.0-

1) '-. w

m

__m upinw w

q

9

3

-

1,- Act ill

FIG. 5. Elevated expression of ERR and H13 genes in tumor cells compared with that in noncancerous cells. Total RNA was prepared from murine noncancerous (normal) thymuses and in vitro-adapted tumor cells of various inbred mice, C57BL/6 and B6.C-H-30c X-irradiation (X-Irr)-induced tumors, and B1O.T(6R) and B10.AQR radiation leukemia virus (RadLV)-induced tumors (A). RNA was also prepared from human noncancerous (normal) tissues resected with malignancy and tumor cell lines including human acute lymphoblastic leukemia (CCRF-CEM), lung carcinoma (A-427), kidney carcinoma (A-498), and colon adenocarcinoma (CaCo-2) (B). After electrophoresis and Northern transfer, murine ERR transcripts (approximately 9 and 8 kb) and human H13 transcript (approximately 9 kb) were detected by hybridization to ERR and H13 cDNA probes, respectively. Equivalent loading and transfer of RNA in all lanes was assessed by hybridization with murine and human I-actin probes.

4380

YOSHIMOTO ET AL.

J . VIROL.

(it) PNIA

( A) l) MS () O

I

I5

4S

96 ir

l)

1

48

96 hr

k1

.r.11-

0

FIG. 6. Decremental decrease of H13 gene expression upon terminal differentiation of HL-60 cells into granulocytes and macrophages. Total RNA was prepared from differentiating HL-60 cells harvested at the indicated times following stimulation with 1.25% dimethyl sulfoxide or 50 ng of PMA per ml (B). Hybridization was carried out using human H13 cDNA (NruI-P.stI) as a probe. The lower panel shows the same blot stripped and subsequently probed with human 13-actin to assess the amount of RNA loaded in each lane.

into granulocytes or macrophages, which are not rapidly proliferating cells, induced by dimethyl sulfoxide or PMA, respectively. After incubation with these agents, morphological changes are observed, and H13 gene expression in both cases decreases gradually to a very low level by 96 h, as shown in Fig. 6. These results may be related to the involvement of H13 in cellular proliferation.

DISCUSSION Both ERR and H13 mRNA are ubiquitously expressed in noncancerous tissues except for the liver (1, 32). This fact is consistent with the result that Moloney murine leukemia virus can infect most murine cells but cannot infect adult hepatocytes (10, 29, 31). However, mouse hepatocytes can be infected during fetal development and liver regeneration. This is presumably because ERR gene expression would be induced by hepatocyte proliferation. In contrast to the ERR and H13 genes, the expression of TEA, which has striking homology to the ERR and H13 genes, is limited to a few cell types such as activated spleen, thymic epithelial, T-lymphoma, and liver cells (18). It should be noted that no other gene except the ERR gene and TEA encoding multiple transmembrane-spanning proteins have been found so far to be induced during T-cell activation (6). T-cell activation following the interaction of T-cell receptor, antigen, and self-histocompatibility molecules initiates the rapid hydrolysis of phosphatidylinositol to diacylglycerol and inositol 1,4,5-triphosphate. Diacylglycerol acts synergistically with calcium and activates PKC, which plays a key role in the cascade of signal transduction, in a process involving translocation of the enzyme from the cytosol to the cell membrane (15). Our results offer evidence that PKC plays a role in increased expression of ERR; (i) PMA enhances ERR gene expression, (ii) PMA analogs lacking the ability to activate PKC fail to enhance ERR gene expression, (iii) the PKC inhibitor H7 inhibits PMA-induced ERR gene expression, and (iv) calcium ionophore induces ERR gene expression. Some proto-oncogenes are regulated during T-cell activa-

tion. The expression of c-myc gene is induced by stimulation with phytohemagglutinin for about 30 min (16, 24). The association of c-myc activation with proliferation is supported by studies showing that antisense oligonucleotides inhibit the activation of the c-myc gene (12). Activation of c-mnyc seems to contribute to the entry of the cells into S

phase during T-cell activation. The expression of ERR is induced by stimulation with several mitogens by about 3 to 6 h. Considering the rapidity of the process and the fact that ERR encodes a cationic amino acid transporter which aids in the passage of essential amino acids such as L-arginine and L-lysine into cells, the ERR gene induction is likely to precede cellular proliferation. Increased transport of these amino acids should be necessary before cellular proliferation can occur. Whether or not the inhibition of ERR gene expression leads to inhibition of cellular proliferation remains to be clarified. Entry of retroviruses into cells involves binding of the viral envelope glycoprotein to a receptor on the cell surface. Viruses using the same receptor can be identified because they exhibit similar host ranges and interfere with each other for entry into cells. Five different murine retrovirus subgroups have been described and are referred to as the ecotropic, amphotropic, xenotropic, polytropic (mink cell focus-forming virus), and IOAl subgroups (25). FV, a polytropic retrovirus, has been demonstrated to bind the ERR through its amino-terminal domain of gp7O envelope glycoprotein (11). The effect of the direct binding of FV to ERR molecules on ERR gene expression is unclear. Gisselbrecht et al. (9) demonstrated that proviral insertion of FV induces a high level of expression of proto-oncogene c-fins mRNA in mouse mycloblastic leukemias. Whether or not the similar mechanism is applicable to FV-induced ERR gene enhancement remains to be elucidated. The results presented here demonstrate that the levels of expression of murine ERR and human H13 genes are highly elevated in rapidly proliferating cells, such as activated T and B lymphocytes, FV-infected cells, and a variety of tumor cells. These results suggest that ERR and H13 genes play an important role in cellular proliferation. The fact that tumor cells appear to require much higher expression of the ERR or H13 protein than noncancerous cells may have wide significance and applicability. ACKNOWLEDGMENTS This work was supported by NIH grants CA31346, CA22247, and CA35482 (to D.M.). REFERENCES 1. Albritton, L. M., L. Tseng, D. Scadden, and J. M. Cunningham. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666. 2. Amari, N. M. B., and D. Meruelo. 1987. Murine thymomas induced by fractionated-X-irradiation have specific T-cell receptor rearrangements and characteristics associated with day-15 to -16 fetal thymocytes. Mol. Cell. Biol. 7:4159-4168. 3. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156-159. 4. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995. 5. Cleveland, D. W., M. A. Lopata, R. J. MacDonald, N. J. Cowan, W. J. Rutter, and M. W. Kirschner. 1980. Number and evolutionary conservation of oL- and P-tubulin and cytoplasmic P- and y-actin genes using specific cloned cDNA probes. Cell 20:95105. 6. Crabtree, G. R. 1989. Contingent genetic regulatory events in T lymphocyte activation. Science 243:355-361. 7. Doolittle, R. F., M. W. Hunkapiller, L. E. Hood, S. G. Devare, K. C. Robbins, S. A. Aaronson, and H. N. Antoniades. 1983. Simian sarcoma virus onc gene, v-sis, is derived from gene (or

VOL. 66, 1992

8. 9.

10.

11.

12.

13.

14.

15.

16. 17. 18.

19.

20.

genes) encoding a platelet-derived growth factor. Science 221: 275-277. Friend, C. 1957. Cell-free transmission in adult swiss mice of a disease having the character of a leukemia. J. Exp. Med. 105:307-318. Gisselbrecht, S., S. Fichelson, B. Sola, D. Bordereaux, A. Hampe, C. Andre, F. Galibert, and P. Tambourin. 1987. Frequent c-fins activation by proviral insertion in mouse myeloblastic leukaemias. Nature (London) 329:259-261. Hatzoglou, M., W. Lamers, F. Bosch, A. Wynshaw-Boris, D. W. Clapp, and R. W. Hanson. 1990. Hepatic gene transfer in animals using retroviruses containing the promoter from the gene for phosphoenolpyruvate carboxykinase. J. Biol. Chem. 265:17285-17293. Heard, J. M., and 0. Danos. 1991. An amino-terminal fragment of the Friend murine leukemia virus envelope glycoprotein binds the ecotropic receptor. J. Virol. 65:4026-4032. Heikkila, R., G. Schwab, E. Wickstrom, S. L. Loke, D. H. Pluznik, R. Watt, and L. M. Neckers. 1987. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from Go to G1. Nature (London) 328:445-449. Hengel, H., B. Allig, H. Wagner, and K. Heeg. 1991. Dissection of signals controlling T cell function and activation: H7, an inhibitor of protein kinase C, blocks induction of primary T cell proliferation by suppressing interleukin (IL) 2 receptor expression without affecting IL2 production. Eur. J. Immunol. 21: 1575-1582. Hidaka, H., M. Inagaki, S. Kawamoto, and Y. Sasaki. 1984. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23:5036-5041. Isakov, N., M. I. Mally, W. Scholz, and A. Altman. 1987. T-lymphocyte activation: the role of protein kinase C and the bifurcating inositol phospholipid signal transduction pathway. Immunol. Rev. 95:89-111. Kelly, K., B. H. Cochran, C. D. Stiles, and P. Leder. 1983. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35:603-610. Kim, J. W., E. I. Closs, L. M. Albritton, and J. M. Cunningham. 1991. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor. Nature (London) 352:725-728. MacLeod, C. L., K. Finley, D. Kakuda, C. A. Kozak, and M. F. Wilkinson. 1990. Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor. Mol. Cell. Biol. 10:3663-3674. Meruelo, D. 1979. A role for elevated H-2 antigen expression in resistance to neoplasia caused by radiation-induced leukemia virus. J. Exp. Med. 149:898-909. Meruelo, D., R. Kornreich, A. Rossomando, C. Pampeno, A. Boral, J. L. Silver, J. Buxbaum, E. H. Weiss, J. J. Devlin, A. L. Mellor, R. A. Flavell, and A. Pellicer. 1986. Lack of class I H-2

ENHANCED GENE EXPRESSION OF ERR AND H13

21.

22.

23. 24.

25. 26.

27.

28. 29.

30.

31.

32.

4381

antigens in cells transformed by radiation leukemia virus is associated with methylation and rearrangement of H-2 DNA. Proc. Natl. Acad. Sci. USA 83:4504-4508. Meruelo, D., M. Lieberman, B. Deak, and H. 0. McDevitt. 1977. Genetic control of radiation leukemia virus-induced tumorigenesis. II. Influence of Srlv-1, a locus not linked to H-2. J. Exp. Med. 146:1088-1095. Meruelo, D., M. Lieberman, N. Ginzton, B. Deak, and H. 0. McDevitt. 1977. Genetic control of radiation leukemia virusinduced tumorigenesis. I. Role of the major murine histocompatibility complex, H-2. J. Exp. Med. 146:1079-1087. Mitchell, R. L., L. Zokas, R. D. Schreiber, and I. M. Verma. 1985. Rapid induction of the expression of proto-oncogene fos during human monocytic differentiation. Cell 40:209-217. Reed, J. C., J. D. Alpers, P. C. Nowell, and R. G. Hoover. 1986. Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc. Natl. Acad. Sci. USA 83:3982-3986. Rein, A., and A. Schultz. 1984. Different recombinant murine leukemia viruses use different cell surface receptors. Virology 136:144-152. Rush, J. S., and C. J. Waechter. 1987. Inhibitors of protein kinase C block activation of B lymphocytes by bacterial lipopolysaccharide. Biochem. Biophys. Res. Commun. 145:13151320. Ullrich, A., L. Coussens, J. S. Hayflick, T. J. Dull, A. Gray, A. W. Tam, J. Lee, Y. Yarden, T. A. Libermann, J. Schlessinger, J. Downward, E. L. V. Mayes, N. Whittle, M. D. Waterfield, and P. H. Seeburg. 1984. Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells. Nature (London) 309:418425. Wang, H., M. P. Kavanaugh, R. A. North, and D. Kabat. 1991. Cell-surface receptor for ecotropic retroviruses is a basic aminoacid transporter. Nature (London) 352:729-731. Weiss, R., N. Teich, H. Varmus, and J. Coffin. 1984. RNA tumor viruses: molecular biology of tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Westin, E. H., F. Wong-Staal, E. P. Gelmann, R. Dalla-Favera, T. S. Papas, J. A. Lautenberger, A. Eva, E. P. Reddy, S. R. Tronick, S. A. Aaronson, and R. C. Gallo. 1982. Expression of cellular homologues of retroviral onc genes in human hematopoietic cells. Proc. Natl. Acad. Sci. USA 79:2490-2494. Wolff, J. A., T. Yee, H. F. Skelly, J. C. Moores, J. G. Respess, T. Friedmann, and H. Leffert. 1987. Expression of retrovirally transduced genes in primary cultures of adult rat hepatocytes. Proc. Natl. Acad. Sci. USA 84:3344-3348. Yoshimoto, T., E. Yoshimoto, and D. Meruelo. 1991. Molecular cloning and characterization of a novel human gene homologous to the murine ecotropic retroviral receptor. Virology 185:10-17.