to more mature myeloid cell forms (metamyelocytes and gran- ulocytes) by using the agents Me2SO and retinoic acid. In ad- dition, K562, a cell line derived from ...
Proc. Nati Acad. Sci. USA Vol. 79, pp. 2194-2198, April 1982
Biochemistry
Differential expression of the amy gene in human hematopoietic cells (onc genes/RNA gel blotting/avian myeloblastosis virus/hematopoietic cell differentiation/HL60-human promyelocytic leukemia cell line)
E. H. WESTIN*, R. C. GALLO*, S. K. ARYA*, A. EVAt, L. M. SOUZAX, M. A. BALUDAt, S. A. AARONSONt AND F. WONG-STAAL* *Laboratory of Tumor Cell Biology, Division of Cancer Treatment, National Cancer Institute and tLaboratory of Cellular and Molecular Biology, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205; *Department of Pathology, University of California, School of Medicine, Los Angeles, California 90024 Communicated by Renato Dulbecco, December 28, 1981
ABSTRACT Total cellular RNAs from a variety of fresh and culture-derived human hematopoietic neoplastic cell types at various stages of differentiation and human sarcoma, carcinoma, melanoma, and glioblastoma cell lines were enriched for poly(A)containing sequences, fractionated by gel electrophoresis, and blot hybridized to a cloned DNA probe containing the transforming sequences (v-amv) of avian myeloblastosis virus (AMV), a virus known to cause myeloid leukemias in chickens. Expression of RNA sequences homologous to AMV was detected in all immature myeloid and lymphoid T cells in addition to the single erythroid cell line examined, but not in mature T cells or in B cells, including lymphoblast cell lines derived from patients with Burkitt lymphoma. In addition, induction of the cell line HL60, a promyelocytic leukemia line, to differentiate with dimethyl sulfoxide or retinoic acid resulted in a reduction of the level of expression of the human cellular gene c-amy homologous to v-amy. There was no detectable c-amv mRNA in any of the solid tumor cell lines examined. Thus, expression of the human c-amy gene could be correlated with the stage ofdifferentiation of different hematopoietic cell types determined by morphologic and marker studies. Expression of c-amy could not be correlated with the extent of methylation in HL60 and in HL60 induced to differentiate with dimethyl sulfoxide.
now convincing evidence that the transforming retroviruses have arisen by recombination ofhelper type-C retroviruses and phylogenetically conserved c-onc genes. The inserted cell-derived genetic information appears necessary for the induction or maintenance, or both, of viral transformation (10, 11). An example of this class of onc genes is the cellular gene homolog of the transforming gene of avian myeloblastosis virus (AMV), which can be found in a variety ofspecies including man (12). AMV causes acute myeloblastic leukemia in chickens and transforms cells of the granulocyte-monocyte lineage (13). These transformed cells have morphologic and marker characteristics of myeloblasts. Although the properties or even existence of the transforming protein of AMV are not known, the presence of one-specific sequences in the AMV genome has been demonstrated with both recycled cDNA (14) and molecularly cloned viral DNA (15). In this study, we examined a wide spectrum of fresh peripheral blood cells and cultured human hematopoietic cells, as well as carcinoma, sarcoma, melanoma, and glioblastoma cell lines, for the expression ofthe cellular amy gene (c-amy) at the mRNA level. The results showed detectable (more than one copy per cell) expression of this gene in precursor cells of the lymphoid, myeloid, and erythroid'series but apparent absence of expression in immature B cells and in more differentiated T cells and myeloid cells. In addition, no solid tumor cell line expressed sequences related to the viral transforming sequences (v-amv).
Human leukemias are monoclonal diseases viewed as specific blockages in hematopoietic cell differentiation. Therefore, fresh leukemic cells and cell lines derived from these may be considered equivalent to clones of normal cells "frozen" at various stages of hematopoiesis. During the past decade, a number of permanent human leukemia and lymphoma cell lines have been established that display stable marker characteristics (for a review, see ref. 1). In addition, a cellline (HL60) established from a patient with acute promyelocytic leukemia (2) can be induced to differentiate to more mature myeloid cells in vitro with dimethyl suffoxide (Me2SO) or retinoic acid (3, 4). These cell lines and fresh leukemic cells can be used to study (i) gene regulation as a function of differentiation and (ii) the mechanism of leukemogenesis. Also, cell lines from many solid tumors, such as carcinomas, sarcomas, glioblastomas, and melanomas, are available to contrast with the hematopoietic cell systems. Among the
MATERIALS AND METHODS
cellular genes of potential interest is the class of genes that has been identified by virtue of its sequence homology to the transforming genes of retroviruses (5-9), many of which cause hematopoietic neoplasias rapidly in vivo. For simplicity, we will refer to these genes as cellular onc genes (c-onc) without implying that these genes themselves are transforming. There is
Cells and Cultures. Permanent human leukemia and lymphoma cell lines that exhibit a stable marker profile of their original tumors and that represent the spectrum of lymphoid, myeloid, and erythroid cell types at various stages in hematopoiesis were selected. A summary of their marker characteristics has been published (1). Cell lines used included the B-cell lines Daudi and Raji (16, 17); immature T-cell lines CCRFCEM, Molt 4, and KM-3 (18-20); the immature myeloid cell line KG-1 (21); and the erythroid precursor cell line K562 (22). HL60, a human promyelocytic cell line (2), was studied in its promyelocytic form and after induction of differentiation with Me2SO and retinoic acid (3, 4). HUT 78 and HUT 102, two mature T-cell lymphoma lines which were established with human T-cell growth factor (23, 24) but which subsequently became independent of T-cell growth factor, presumably because of autonomous production of the growth factor (25, 26), were
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Abbreviations: AMV, avian myeloblastosis virus; AML, acute myelogenous leukemia; ALL, acute lymphocytic leukemia; kb, kilobase(s); Me2SO, dimethyl sulfoxide. 2194
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Westin et aL
also studied. Fresh leukemic cells were obtained from patients with acute myelogenous (AML) and acute lymphoid leukemias (ALL) by using a cell separator. Solid tumor cell lines derived from a variety of carcinomas, sarcomas, melanomas, and glioblastomas were also studied. The type of tumor from which these cell lines were derived is shown in Table 1. They include AlOlD, A172, A388, A375, A431, A498, A549, and A673 (27); HOS and 8387 (28, 29); and A1235, A1306, A1383, A1604, A1617, A2394, A2780, and A7095 (S. A. Aaronson and N. W. Ellmore, unpublished data). Preparation of RNA. Total cellular RNA was extracted by using a modified guanidine hydrochloride extraction technique (30, 31). Briefly, 109 cells were pelleted and solubilized in 10 ml of buffer A [8 M guanidine hydrochloride/10 mM sodium acetate, pH.5.0] and homogenized with 20-30 strokes of a loosefitting pestle in a Dounce homogenizer. RNA was precipitated with 1/2 volume of ethanol at -20TC for 30 min. After centrifugation, the RNA pellet was dissolved (in a decreasing volume ofbuffer A with 20 mM EDTA, pH 7.5) and reprecipitated (with 1/2 volume of ethanol) three additional times. The final precipitate was redissolved in 5 ml of 20 mM EDTA (pH 7.5) and extracted with an equal volume of chloroform/isobutanol (4:1). The RNA then was precipitated overnight at -20°C in 3 M sodium acetate (pH 6.0) and then reprecipitated in 0.2 M sodium acetate (pH 5.0) with 2 volumes of ethanol at -20°C. The final RNA precipitate was redissolved in water, and total cellular poly(A)-containing RNA was further enriched by affinity chromatography with oligo(dT)-cellulose (32). RNA Gel Blotting Procedure. Five micrograms of poly(A)selected RNA per lane was electrophoresed in 1% agarose gels containing 5 mM methyl mercury, transferred to nitrocellulose with 3.0 M sodium chloride/0.3 M sodium citrate, pH 7, and hybridized as described by Thomas (33), except that the competitor used to prevent absorption of nonspecific counts during hybridization was Escherichia coli DNA (250 ,ug/ml) rather than salmon sperm DNA. Filter-washing conditions were made less stringent than described (33) by washing four times in 0.3 M sodium chloride/0.03 M sodium citrate, pH 7/0.1% NaDodSO4 at room temperature for 5 min each, then two times in 0.15 M sodium chloride/0.015 M sodium citrate, pH 7/ 0.1% NaDodSO4 at 42°C for 15 min each. DNA Gel Blotting Procedure. To study methylation of the c-amv DNA locus as a function of RNA expression, we digested DNA derived from undifferentiated and differentiated HL60 cells (induced with Me2SO) with the restriction endonucleases Hpa II and Msp I according to conditions recommended by the manufacturer (New England BioLabs). Fragments were then separated on 1% agarose gels, transferred to nitrocellulose, and hybridized according to the procedure of Southern (34). Derivation of amv Probe and Nick Translation. The cloning of the integrated genome of AMV has been described (15). A subclone of a HindlIl-HindIlI fragment in pBR322 containing the entire amv gene and some helper virus-derived sequences was nick translated as described (35) with specific activities of 2-4 X 108 cpm/,ug of input DNA per ml.
RESULTS Expression of c-amv in Human Leukemic Myeloid and Erythroid Cells. Poly(A)-containing RNA was prepared from fresh peripheral blood leukocytes of five patients with AML and from the cell lines KG-1, a myeloid precursor line, and HL60, apromyelocytic cell line. HL6O was also induced to differentiate to more mature myeloid cell forms (metamyelocytes and granulocytes) by using the agents Me2SO and retinoic acid. In addition, K562, a cell line derived from a patient with chronic
Proc. NatL Acad. Sci. USA 79 (1982)
2195
myelogenous leukemia but which has some erythroid precursor characteristics (36, 37), was examined. The RNA samples were fractionated on methyl mercury gels, blotted onto nitrocellulose filters, and hybridized to a [32P]DNA fragment containing amv sequences. A single mRNA species of 4.5 kilobases (kb) was detected in all cells examined. However, in HL60 cells induced to differentiate with either Me2SO or retinoic acid, the level of RNA from the human c-amv gene was drastically diminished (Fig. la). Induction of differentiation was not complete with immature cells constituting 20% of the cell population, and the residual band seen is presumably due to these cells. In a related study (38), we have found that with the acute leukemia virus MC-29, which has a much higher level of expression in HL60, the residual level of its oncogene c-myc-related RNA is correlated with the number ofimmature cells present after induction of differentiation. The decrease in expression of these sequences is not due to nonspecific degradation of the RNA because the amount of transcripts of the gene related to the onc gene of Abelson murine leukemia virus was not altered (Fig. lb). Lack of Correlation of Extent of DNA Methylation with Expression of c-amv. Recently, attention has been focused on the possible role of extent of methylation of DNA as a controlling factor in expression of various RNA's (38-41). In order to examine this in the case ofc-amv expression, DNA's were isolated from HL60 and from HL60 induced to differentiate with Me2SO. These were digested with the restriction endonucleases Hpa II and Msp I, which recognize the same four-base sequence C-C-G-G except that Msp I but not Hpa II will cleave the sequence if a methylated-C residue is present. The c-amv locus of HL60 with or without induction is highly methylated because Hpa II digestion yielded two bands of high molecular weight (10.5 and 8.3 kb), whereas Msp I-digested DNA yielded bands of much lower molecular weight (3.7 and 3.4 kb) (Fig. 2). Thus, the extent of DNA methylation of c-amv assayed by this technique appeared unchanged regardless of whether this gene was transcriptionally active as in undifferentiated HL60 or inactive as in differentiated HL60 cells. Expression of c-amv in Human Lymphoid Cells. Many human cell lines of the lymphoid (T, B, and null) series are available. We have examined the following cell lines in this study: KM-3, a leukemic non-T- and non-B-cell line exhibiting marker antigens of lymphoid precursor cells; Daudi and Raji, lymphoblast B-cell lines derived from patients with Burkitt lymphoma; a
b
1
I 1
4.5 kb,
I,
2
3
"A".t,:"& R
.z,
4
5
I
I 1
2
W- .10
;,..-4 i. "
VI..
FIG. 1. Hybridization of v-amv to myeloid and erythroid cells. (a) Hybridization of v-amv to RNA from HL60 (lane 1), HL60 induced to differentiate with Me2SO (lane 2), fresh peripheral blood leukemic myeloblasts from a patient with AML (lane 3), KG-1 (lane 4), and K562 (lane 5). (b) Hybridization of v-abl (Abelson murine leukemia virus onc-derived sequences) to RNA from HL60 (lane 1) and from HL60 induced to differentiate with Me2SO (lane 2). Multiple bands are visualized with v-abl, with the dominant bands being 7.2 and 6.4 kb.
Proc. Nad Acad. Sci. USA 79 (1982)
Biochemistry: Westin et aL
2196
Table 1. Summary of fresh human cells and cell lines tested for
b I
I
I
expression of c-amv
-
2
'1
2
Cell type Hematopoietic cells Myeloid
Erythroid Lymphoid T cells
B cells
Expressing
Not expressing
KG-1 HL60 Fresh peripheral blood myeloblasts* K562 CEM Molt 4 KM-3 Fresh T-cell ALL blastst
Hut 78 Hut 102
Daudi
Raji Solid tumors Rhabdomyosarcoma Osteogenic sarcoma Fibrosarcoma FIG.
Extent
2.
of
methylation
of
c-amy
in'HL6
and
in
HL60
in-
dimethyl sulfoxide. (a) Hpa H1 restriction digests of HLIM (lane 1) and HLIM induced with Me2SO (lane 2). The size of the bands are 10.5 and, 8.3 kb. (b) Msp I -restriction digests of HL6O (lane 1) and HL60 induced with Me2SO (lane 2). The size of the
duced to differentiate with-
bands
are
3.7 and 3.4 kb.
GEM, terminal deoxyribonucleotidyltransferasepositive immature T-cell lines derived from patients with ALL; and HUT 102 and HUT 78, terminal deoxynibonucleotidyltransMolt 4 and
T-cell lines established from patients T-cell lymphomas. HUT 102 also produces a
ferase-negative
mature
with cutaneous
(human T-cell lymphoma virus) (24). sample from a patient with T-cell ALL (erythrocyte rosette positive) was also analyzed. Fig. 3 shows results of hybridization with RNAs from cells in this category. The same 4.5-kb band detected in RNA from the myeloid cells was de-
human retrovirus, HTLV
Synovial sarcoma Skin carcinoma Lung carcinoma Gastrointestinal carcinoma Renal carcinoma Bladder carcinoma Ovarian carcinoma Melanoma
Glioblastoma
One fresh cell
tected in RNA from the cell lines KM-3, T-cell ALL. In contrast, this -transcript
-102 and
HUT 78
lines tested sion of
RNA
GEM, and Molt 4 and
peripheral blood leukocytes
from the fresh
(mature
T
cells)
nor
was
from
in the
a
patient with
detected in HUT
not
lymphoblast
immature T cells
was
reproducible
with several
preparations.
Absence of Expression of c-amy in Human Solid Tumor Cell Lines. Numerous cell lines derived from human solid tumors are
available. To
study expression
from cell lines of
1
4.5 kb
a
variety of
2
*-PRw
-
_
3
w
of c-amy,
tumor
4
we
analyzed
RNA
types (Table 1). Blot hy-
5
6
i
n..
.,.z-:,.,
a
8
7
.,-id
A1617 A498 A1604 A2780
AlOlD A375 A1306 A172 A1235
* Five patients. t One patient.
bridization with poly(A)-selected RNAs from these cell lines showed no detectable expression of c-amv.
B-cell
(Daiudi and Raji).The relatively high. level of expres-
c-amv
A673 HOS A2394 8387 A1383 A1095 A388 A431 A549
'%
"..l. Alawl",
FIG. 3. Hybridization. of v-amv to lymphoid cells. Lanes contain RNA from: 1, Molt 4; 2, CEM; 3, KM-3; 4, fresh peripheral blood lymphoblasts from a patient with ALL {erythrocyte rosette positive; terminal deoxyribonucleotidyltransferase negative); 5, Daudi; 6, Raji; 7, HUT 102; and 8, HUT 78.
DISCUSSION We demonstrated that the human gene analog of the transforming gene of AMV is transcribed in certain hematopoietic cells as a single mRNA species of 4.5 kb. In HL60, extent of methylation could not be correlated withtthe extent of expression of c-amv. Attempts to detect expression of c-amv in a large number of human solid tumors and normal fibroblast cell lines have failed. Thus, c-amv seems to be expressed specifically in hematopoietic cells. In hematopoiesis, a single pluripotential stem cell is thought to generate a proliferating cell pool with subsequent differentiation toward four pathways-i.e., T and B lymphocytes, granulocytes, and erythrocytes. From the analysis of cell lines, it appears that lymphoid T and B cells share a common precursor cell after commitment to lymphoid differentiation (1, 42). Each pathway is probably represented by a continuum of maturing cells but can be subdivided into several compartments based on marker profiles. A study of 50 human hematopoietic cell lines using many cell surface and enzymatic markers, as well as a series of monoclonal. antibody profiles, has led Minowada et aL (1) to place various cell lines at precise steps in the differentiation scheme of hematopoiesis (Fig. 4). For example, in
Biochemistry:
Westin et al.
Proc. Natl.Acad. Sci. USA 79 (1982)
2197
w T-ALL
Sm
B-ALL
S
w5|
*-$I
RAN CYTE HL6O + DMSO OR
AML
lS
FIG. 4. Summary of expression of c-amv in human myeloid, erythroid, and lymphoid cells. Schema was adapted from that proposed by Minowada et al. (1) based on cell surface marker studies of numerous human leukemia and lymphoma cell lines. 0, Cell types found to be expressing c-amy; 0, cells without detectable levels of c-amy mRNA; 0, areas in the schema not examined.
the T-cell lymphocyte differentiation pathway, CEM and Molt 4 represent immature cells (T-cell blasts), whereas the terminal deoxyribonucleotidyltransferase-negative cell lines HUT 102 and HUT 78 represent mature T cells. Cells of the myeloid lineage have characteristics of myeloblasts, promyelocytes, myelocytes, metamyelocytes, and granulocytes as they proceed in differentiation. The erythroid pathway is the most poorly defined in terms of available cell lines and so far has only one known candidate member, an erythroblast-like cell line, K562 (36, 37). Fresh leukemic cells also can be typed and placed in this scheme according to their morphological characteristics and marker profiles. Myeloblasts are represented by fresh AML cells, and immature T-cell lymphoblasts are represented by ALL T-cells. The fresh leukemic cell samples we studied fall into these two categories. The c-amv gene is transcribed in precursor cells of lymphoid, myeloid, and erythroid lineages (Fig. 4). Transcription of c-amy was not demonstrated throughout human B-cell differentiation nor in late T-cell and myeloid cell differentiation. Terminally differentiated HL60 cells, terminal deoxyribonucleotidyltransferase-negative mature T cells (HUT 102 and HUT 78), in addition to several lymphoblast B-cell lines failed to express detectable levels of c-amy mRNA. Thus, our findings indicate that the c-amy gene may play a role in early human hematopoietic cell differentiation. However, we should point out that the sensitivity of the assay system used is approximately one RNA copy per cell using a homologous DNA probe (43) and, therefore, will be less sensitive in the present case in which heterologous viral-derived amy sequences are used as the probe. Thus, failure to detect expression would not rule out extremely low levels of c-amy expression. Although AMV induces myelogenous leukemias in chickens and transforms chicken myeloblasts in vitro (44), it is not known whether the human c-amy sequences are involved in human
leukemias. Several studies have implicated derepression ofcellular onc genes in retrovirus-induced tumorigenesis. In B-cell lymphomas of chickens induced by two distinct viruses, avian leukosis virus and avian syncytia virus, the exogenous proviruses are integrated in the vicinity of c-myc and, as a result, expression of c-myc is greatly enhanced (45-48). Less frequently, avian leukosis virus can induce erythroblastosis, and, in these tumors, the avian leukosis provirus has been shown to integrate next to c-erb, a cellular gene homologous to the transforming gene of avian erythroblastosis virus (H. J. Kung, personal communication). However, there is as yet no evidence for activation of onc genes in nonviral induced tumors in animal systems. The various myeloid cell lines and fresh human AML cell samples examined in this study did not express inordinately high levels of c-amy transcript compared to the lymphoblast T-cell lines. Our present findings are compatible with the possibility that the c-amy gene is activated only during particular stages of normal hemnatopoietic cell differentiation. Whether or not it also plays a role in transformation of certain human hematopoietic cells remains to be determined. We would like to thank Dr. Carlo Moscovici for a gift of AMV-transformed nonproducer cells, Karen Gorse for excellent technical assistance, and Anna Mazzuca for invaluable help in preparation of this
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