Oncogene expression in T-cell lymphoproliferative disorders

Leukemia Research Vol. 12, No. 4, pp. 327-337, 1988. Printed in Great Britain.

0145-2126/88 $3.00 + .00 Pergamon Press plc

O N C O G E N E EXPRESSION IN T-CELL L Y M P H O P R O L I F E R A T I V E DISORDERS GAYLE E. WOLOSCHAK,* W. CRAIG HOOPER,* MARY J. DOERGE,* ROBERT L. PHYLIKY,t THOMAS E. WITZIG,t PETER M. BANKS,~:GORDON W. DEWALD§ and CHIN-YANG LI]I * Dept of Immunology and Dept of Biochemistry and Molecular Biology, ? Division of Hematology, $ Dept of Pathology, § Cytogenetics Laboratory and II Department Laboratory Hematology, Mayo Clinic and Foundation, Rochester, MN 55905, U.S.A.

(Received 14 September 1987. Revision accepted 2 January 1988) Abstract--We have investigated the expression of oncogenes and other related genes in eleven patients with T-cell lymphoproliferative disorders and ten patients with other hematologic malignancies. The phenotypes of the T-cell disorders were determined using monoclonal antibodies specific for helper or suppressor subsets. RNA preparations were isolated from peripheral blood mononuclear cells and/ or lymph node sections, 5'-end labeled with 7-32p-ATP, and hybridized under stringent conditions to an excess of nitrocellulose-bound specific cloned DNA; autoradiographs were analysed by microdensitometry. Results revealed increased expression of K-ras, v-fps, transferrin receptor, ol-tubulin and oL-interferonin at least five of six helper T-cell lymphoproliferative disorders, while five of five suppressor T-cell disorders demonstrated levels of hybridization to these clones no higher than background. However, studies of T-suppressor disorders demonstrated enhanced levels of r-interferon-specific RNA in five of five patients, an increase apparent in three of six T-helper chronic lymphoproliferative disorders. These results demonstrate different patterns of gene expression evident in T-helper and T-suppressor abnormalities.

Key words: Oncogenes, leukemia, lymphoma, T-cells, lymphoproliferation, gene expression, interferon. myelogenous leukemia (AML) or acute lymphocytic leukemia (ALL) had increased accumulation of cmyb RNA, whereas cells from patients with chronic myelogenous leukemia (CML) or B-cell chronic lymphocytic leukemia (B-CLL) did not. c-myc R N A was variably expressed in cells of patients with AML, ALL, or CML but not B-CLL. They were unable to demonstrate DNA rearrangement or amplification of c-myb or c-myc in any sample. Rosson et al. [7] studied cells from 18 patients with either acute or chronic leukemia and found c-myc RNA to be expressed in all patients with no pattern of specificity. c-myb appeared to be expressed only in cells from one AML patient, and the only two patients in which erb B was elevated had AML. Although investigations of cells from other patients with leukemia demonstrated variable elevations in mRNA for erb B, myc, myb, fes, fps, and src compared to background controls [8-10], no consistent pattern of mRNA expression was associated with a specific type of leukemia. In our studies, the level of expression of oncogenes and other growth-related genes was measured in cells from six patients with helper T-cell (TH) and five patients with suppressor T-cell (Ts) lymphoproliferative disorders.

INTRODUCTION RECENT evidence has demonstrated that mutation, gene rearrangement and gene amplification can all lead to altered transcription of specific cellular oncogene sequences [1-3]. However, many tumors display altered mRNA transcription and mRNA accumulation in the absence of detectable changes at the DNA level [4, 5]. The mechanisms underlying these alterations in expression are unknown, and the oncogenic role played by abnormal amounts of oncogenespecific mRNA is unknown. Cells from many hematologic malignancies express enhanced levels of oncogene-specific mRNA. In some studies differences in mRNA expression have been found between related disease entities. Ferrari et al. [6] found that cells from patients with acute

Abbreviations: PBL, peripheral blood lymphocytes; CLL, chronic lymphocytic leukemia; TH, helper T-cell; Ts, suppressor T-cell; NHL, non-Hodgkin's lymphoma; ND, not done; HND, hybridization not detected; IL2, interleukin 2. Correspondence to: Dr G. E. Woloschak, Division of Biological, Environmental and Medical Research, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, U.S.A. 327

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G . E . WOLOSCHAKet al.

In one type of T-cell malignancy, T-CLL, it has recently b e c o m e apparent that the surface m e m b r a n e p h e n o t y p e is an important predictor of clinical course and disease activity. Patients with TH-CLL usually have an aggressive disease course requiring early cytotoxic treatment with a median survival of only 21 months [11], whereas patients with Ts-CLL have an indolent disease course and may survive years without any cytotoxic therapy [12]. Because of these important clinical features, we were interested in determining any differences in level or pattern of m R N A expression between these two T-cell subsets. We also studied 10 additional patients with other hematologic malignancies. MATERIALS AND METHODS

cDNA probes pAM91 actin-specific cDNA probe was obtained from Dr A. Minty [13]. c-H-ras, c-K-ras, c-mos, v-src, N-myc, v-erb B, c-myc, N-ras, v-fos and v-fins probes were obtained from the American Type Culture Collection. Transferrin receptor was provided by Dr F. Ruddle [14], IL2 and ~interferon by Dr Taniguchi [15, 16], a~-interferon by Dr C. Weissmann [17], v-s/s by Dr R. Gallo [18], o:tubulin by Dr C. Veneziale at Mayo Clinic, v-fps and vabl by Dr A. Balmain, v-tel by Dr H. Temin [19], v-bas and v-myb by Dr R. Scott at Mayo Clinic, v-fes by Dr C. J. Sherr [20] and p53 by Dr A. Levine [21]. Cytogenetics Chromosome studies were done on peripheral blood lymphocytes (PBL), bone marrow or lymph node cell preparations. Each slide was prepared for chromosome analysis using a direct technique, short-term unstimulated cultures or both [22]. Slide preparations were stained for chromosome analysis by GTG-banding or QFQ-banding or both of these methods [23]. In each case, ten to forty metaphases were examined. Determinations of cell surface phenotype The cell surface phenotype was determined by use of monoclonal antibody (MoAb) reagents from the Leu (Becton-Dickinson MoAb Center, Inc., Mountain View, CA), OK (Ortho Diagnostic Systems, Inc., Raritan, NJ), and Lyt (New England Nuclear, Boston, MA) series, as well as the B1 antibody (Coulter, Hialeah, FL). When the cells were obtained from peripheral blood, air-dried smears were prepared and immunotyping performed using an indirect immunocytochemical technique with alkaline phosphatase as the indicator [24]. Lymph node specimens were immunotyped by both an immunoperoxidase method on frozen section [25] and by FACS IV flow cytometry (Becton-Dickinson) using fluorescein isothiocyanate conjugated MoAb in single cell suspensions. Dot blot hybridizations RNA derived from tissue samples or ficoll-gradient separated PBL was purified in three steps, (1) alkaline phenol extraction (2) precipitation from 3M sodium acetate and (3) oligo dT cellulose column chromatography [26-29]. Standard dot blot hybridizations were used to obtain relative estimates of the amounts of specific RNAs within

each preparation. The technique of dotting excess clonespecific DNA and hybridizing to 32P-labeled RNA has been used extensively by ourselves [27-29] and others [26] as a means of measuring relative quantities of specific RNAs in a single preparation. In all experiments, 1 ~tg doublestranded cloned plasmid DNA (determined to be DNAexcess for the reaction) in 1 M ammonium acetate was dotted onto a nitrocellulose filter. Filters were washed in 3 × SSC (45 mM Na citrate, pH 7.4, 0.45 M NaCI) and baked in a vacuum oven at 80°C overnight. Prior to hybridization, filters were soaked in i x Denhardt's solution (0.2% ficoll, 0.2% bovine serum albumin, 0.2% polyvinylpyrrolidine), 3 × SSC at room temperature for 1 h followed by washing for 1 h at 65°C in hybridization buffer (50% formamide, 1 × Denhardt's solution, 10 ~g/ml Poly A, 50 ~tg/ml herring sperm DNA, 3 × SSC). RNA was 5'-end labeled with 32p as follows. 2 ptg RNA were partially hydrolyzed with NaOH. After neutralization of the NaOH with HC1, the RNA was incubated at 37°C for 45 min with T4 polynucleotide kinase and 50 ktCi ),_32p_ labeled ATP (3000 Ci/mmol.). RNA was separated from unincorporated ATP by Sephadex G50 column chromatography at room temperature in 3 × SSC. Prior to hybridization, RNA aggregates were broken up by heat-shocking the sample for 1 min at 90°C. The RNA was hybridized to the nitrocellulose filters dotted with DNA probes at 50°C for 18 h. Filters were washed three times for 1 h each in 3 × SSC at 65°C and three times for 1 h each in 0.1 × SSC at 65°C. Nitrocellulose filters were set up for autoradiography with X-ray film at -70°C for 24 h. After autoradiographs were obtained, individual dots were removed and densitometric analysis was performed. All reported results were based on hybridization of 5 × 107 counts/min total counts and exposure of blots for 96h. From patient samples, sufficient RNA was obtained to perform experiments twice; therefore, Tables 3--5 are based on duplicate experiments done independently. RESULTS Clinical data Table I outlines clinical and pathologic data on the 21 patients in this study. Six patients were diagnosed as having T-helper cell lymphoproliferative disorders, while five patients had T-lymphoproliferative disorders of the suppressor type. Ten additional patients with various hematologic malignancies (one patient with Hairy cell leukemia, four with B-cell non-Hodgkin's l y m p h o m a , one with H o d g k i n ' s disease, two with B - C L L and one with t h y m o m a ) were used as controls. For proliferationspecific controls, lymph nodes and tonsils from normal individuals as well as PHA-activated P B L were used. As can be seen in Table 2, no consistent c h r o m o s o m a l abnormalities were found a m o n g these patients or when comparing T-helper and T-suppressor disorders.

Gene expression studies R N A derived from P B L or t u m o r tissue from each of the patients was labeled in a kinase reaction and hybridized to nitrocellulose-bound cloned D N A s

Oncogenes and T-iymphoproliferative disorders

329

TABLE 1. PATHOLOGICAND CELLSURFACEPHENOTYPERESULTSON PATIENTS Patient No.

Age

Sex

1

68

F

2

51

F

3

28

F

4 5 6

69 69 69

F M F

7 8 9 10

84 37 48 58

M M M M

11

65

M

12 13

72 19

M F

14

43

F

15

72

M

16

72

M

17 18 19

60 55 67

Misc. 20 21

Pathologic diagnosis

Cell source

Cellular phenotype

T-helper lymphoproliferative disorders diffuse, mixed NHL* to thyroid with peripheral blood involvement diffuse, mixed NHL

PBLt

Leu 4+ OKT 4+

fight lower pulmonary nodule PBL

Leu 4+ Leu 3+ Leu 4+, OKT 4+

right pulmonary nodule paratracheal lymph node cervical lymph node

Leu 1+, Leu 3+ Leu 1+, Leu 3+ Leu 4+, Leu 3+

Ts-CLL~ Ts-CLL Ts-lymphoeytosis Ts-CLL, aberrant phenotype diffuse large-cell (immunoblastic) NHL

PBL PBL PBL PBL

Leu 4+, Lyt 3+, Leu 4+, Lyt 3+,

right groin lymph node

Leu 1+, Leu 2+

spleen; PBL omental lymph node

TRAP+§ K+, Leu 14+

fight axial lymph node

K-, ).+

jejunal lymph node

;t+, B I +

PBL

;t÷, B I +

F F M

hairy cell leukemia diffuse small noncleaved cell (pleomorphic) NHL diffuse large-cell (immunoblastic) NHL follicular, small cleaved cell NHL diffuse small lymphocytic NHL B-CLL B-CLL diffuse, mixed NHL

PBL PBL lymph node

DR+ D R + , K+ ).+, B I +

57

F

thymoma

thymus

NDII

15

F

Hodgkin's disease. nodular, sclerosingtype

lymph node

ND

T-helper leukemia/ lymphoma diffuse, mixed NHL diffuse, mixed NHL diffuse, mixed NHL

T-suppressor lymphoproliferative disorders OKT 8+ OKT 8+ OKT 8+ OKT 8+

B-cell lymphoproliferative disorders

* NHL = non-Hodgkin's lymphoma. t PBL = peripheral blood lymphocytes. CLL = chronic lymphocytic leukemia. § TRAP = tartrate resistant acid phosphatase. [[ ND = not done.

G. E. WOLOSCHAK et al.

330

TABLE 2. CYTOGENETICS RESULTS ON PATIENTS TESTED

Patient No.

Source of tissue for chromosome studies

1

lymph node

2 3

right lung lymph node bone marrow

8 9 10 13

peripheral blood peripheral blood peripheral blood lymph node

19

lymph node

present in vast excess. The resulting autoradiographs (Fig. 1) were analysed by microdensitometry to determine relative levels of gene-specific m R N A in each of the cells. Controls of tonsillar tissue, normal lymph nodes, resting and PHA-activated (for 48 h) PBL all exhibited low levels of hybridization to actin, tr-tubulin and sis. The levels of hybridization (as measured by microdensitometry) were only slightly higher than to control parent plasmid (pBR322 and p A C Y C ) . Longer exposures of the blots (7-14 days)

Chromosome analysis 13=47,X,- X , - 5 , - 10,[del(5)(q13q33) =2] marl1, del(15)(q13),t(16;?)(q13;?) +mar, tr(?)[AN] 30=46,XX[NN] 17 =46,XX,t(7;?)(p2?2;?),t(4;9) (q21;p24),mar10,t(17;?)(p1?l;?), t(18;?)(q2?2;?),t(19;?)(pla;?)[gN] 32=46,XY[NN] 20=46,XY,inv(9)(pllq13)[NN] 28 =46,XY,var(Y)(q12,QFQ15)[NN] 20=38-54,XX,+?X, + 12, + 17, + 21, + 2 - 5mar[AA] 1= 46,XY/19 =45 - 47,X,-Y, +3,del(6)(q21q25), t(14;?)(q32;?),+mar6,+ 1-2mar[AN]

revealed increases in m R N A for transferrin receptor in activated lymphocytes (data not shown). Table 3 presents results of experiments aimed at determining relative amounts of gene-specific m R N A in T-helper lymphoproliferative disorders. Microdensitometric analyses were set up in a 1+ to 4 + format (see footnotes to Table 3). Interestingly, cells from all six patients contained elevated levels of R N A specific for transferrin receptor, tr-tubulin, a~interferon and fps. In addition, enhanced accumu-

TABLE 3. RELATIVEmRNA ACCUMULATION IN T-HELPER DISORDERS* Probe Actin Transferrin receptor o~-tubulin H-ras K-ras tr-interferon fl-interferon

fes fps pBR322, s/s erb B

myb los p53

rel bas IL2 receptor§ N-ras

Control

No. 1

No. 2

No. 3

No. 4

No. 5

No. 6

HND t HND HND HND HND HND HND HND HND HND + HND HND HND HND HND HND HND HND

HND + + HND + 2+ HND 2+ + HND HND HND HND HND HND HND HND NTII NT

4+ 3+ 2+ HND + 2+ 2+ HND + HND HND HND HND HND HND HND HND HND HND

3+ + + HND + 3+ + HND 2+ HND HND HND HND HND HND HND lIND NT NT

+ + + + + + + HND + HND HND + HND + HND HND HND NT NT

+ + HND + HND + HND HND + HND HND HND + + HND HND HND HND HND

4+ 4+ 2+ + HND + HND HND 2+ HND HND + HND HND 4+ 4+ 2+ 3+ HND

* Microdensitometric results were quantitated and set up on a + to 4+ scale as follows: 1+ =<25,000 units; 2+=25,001-50,000 units; 3+=50,001-100,000 units; 4+=>100,001 units. t HND = hybridization not detected; mRNA specific for the following clones could not be detected in any of the samples: myc, mos, abl, N-myc, raf. :~ pBR322 = control plasmid. § IL2 = interleukin 2. IINT = not tested.

Control T# -CLL

~-Lymh~a

B-CLL

Actin Transferrin

receptor

o(-tubulin K-ras H-ras cX-interferon ,8 -interferon fes fps pBR322

FIG. 1. Representative RNA slot blot depicting relative hybridization in control (tonsiUar tissue), T-helper (TH)CLL (patient No. 1), T-helper lymphoma (patient No. 3), T-helper lymphoma (patient No. 2), B-cell CLL (patient No. 18), and T-suppressor (Ts)CLL (patient No. 8). In these experiments RNA derived from tumor cells was labelled with y-aEp-ATP in a kinase reaction and hybridized to cloned DNAs immobilized on nitrocellulose filters. Equal numbers of counts of labelled RNA were hybridized in each lane.

331

"Is -CLL

Oncogenes and T-lymphoproliferative disorders

lation of mRNA specific for K-ras and actin was evident in five of the six mRNA samples tested. Each sample contained high levels (relative to controls) of mRNA specific for at least three different cellular oncogenes. In addition, comparisons of Tables 2 and 3 reveal no relationship between specific chromosomal abnormalities and specific gene induction, although more oncogenes were expressed in patients bearing clones with multiple chromosomal abnormalities. Similar experiments performed with four chronic lymphoproliferative disorders involving Tsuppressor cells and in one high-grade lymphoma expressing T-suppressor phenotype demonstrated only fl-interferon to be commonly expressed in increased amounts. Genes expressed at high levels in T-helper disorders were not detectably elevated in T-suppressor diseases. Autoradiographs representative of T-helper and T-suppressor lymphoproliferative disorders are evident in Fig. 1. It is also interesting to note that the patterns of gene expression detected here were stable with time. Three independent samples of blood drawn from patient No. 8 at intervals of three months were used in RNA preparations. These studies always revealed an identical pattern of gene expression. Several other hematologic diseases were studied for gene expression to determine whether patterns evident in T-cell disorders were commonly found in other malignancies (Table 5). It is clearly evident that most genes expressed in T-cell lymphoproliferative disorders are also expressed commonly in one or several other hematologic malignancies. It is interesting to note the absence of a common pattern of gene expression in B-cell malignancies. In addition, as in T-ceU disorders, there were no correlations

333

between chromosomal abnormalities and the chromosomal locations of oncogenes shown to be elevated in expression.

DISCUSSION We examined the expression of a variety of oncogenes and other growth-related genes in eleven patients with T-cell lymphoproliferative disorders and ten patients with other lymphoid malignancies. There was an elevation in expression of oncogenespecific mRNA (K-ras, fps) as well as other RNAs (transferrin receptor, a~-tubulin) in cells of patients with T-helper but not with T-suppressor disorders. It is possible that the enhanced expression of growthspecific genes and oncogenes found in the TH disorder is related to a higher proliferative rate of these cells; however, results from both PHA-activated peripheral blood lymphocytes and inflammatory tonsillar tissues used as controls in this study did not reveal similar patterns of gene expression. This would indicate that mRNA expression may be in part independent of proliferation. We compared the results of chromosome analyses with those of oncogene expression and found no apparent correlation. This was disappointing because in many lymphoid tumor systems there are examples of enhanced oncogene expression that correlate with chromosomal translocation, gene amplification or both or these anomalies [29-31]. A chromosomally abnormal clone was found in only four patients, and in each of these cases there were many chromosome abnormalities. The complex nature of these abnormal clones may be confounding the results of molecular analysis. None of our patients had a clone of

TABLE 4. RELATIVEm R N A ACCUMULATIONIN T-SuPPRESSOR DISORDERS*

Probe fl-interferon fes pBR3221°, s/s myb IL2 receptor§ N-ras

Control

No. 7

No. 8

No. 9

No. 10

No. 11

HNDt HND HND + HND HND HND

+ HND HND + HND NTI[ NT

+ HND HND + HND NT NT

+ + HND + HND NT NT

+ HND HND HND HND HND HND

+ HND HND HND + HND HND

* Microdensitometric results were quantitated and set up on a + to 4+ scale as follows: 1+=<25,000 units; 2+=25,001-50,000 units, 3+=50,0001-100,000 units; 4+=>100,001 units. t HND = hybridization not detected; mRNA specific for the following clones could not be detected in any samples: actin, transferrin, receptor, o:-tubulin, H-ras, K-ras, o-interferon, fps, erb B, myc, mos, p53, tel, bas, abl, N-myc, raf. $ pBR322 = control plasmid. § IL2 = interleukin 2. IINT = not tested.

HND 2+ HND HND HND 3+ + HND HND HND + HND HND HND HND HND 3+ + 3+ HND

HNDt HND HND HND HND HND HND HND HND + HND HND HND HND HND HND HND HND HND HND

+ HND HND HND HND HND HND lIND HND 2+ 2+ HND HND + HND HND HND HND HND

HND HND HND lIND HND HND HND HND HND HND HND + HND HND + + NTII HND HND HND

HND 2+ 2+ + 3+ 2+ HND + HND HND HND HND HND HND HND HND NT HND HND NT

3+ HND HND + HND + lIND HND HND + HND HND + HND HND HND NT HND HND NT

+ HND HND HND HND HND HND + HND HND HND HND 2+ HND HND HND NT HND HND NT

HND HND HND HND HND + HND HND HND HND HND HND HND HND HND HND NT HND HND NT

+ + HND HND + HND HND HND HND + HND + HND HND HND HND NT HND HND NT

+

+ HND HND HND + HND HND HND HND + HND HND HND HND HND HND HND HND HND

HND

No. 20 thymoma

+ HND + + + HND HND HND 2+ HND + + + HND HND NT HND HND NT

+

No. 21 Hodgkin's disease

$ pBR322 = control plasmid. § IL2 = interleukin 2. II NT = not tested.

rel.

* Microdensitometric results were quantitated and set up on a + to 4 + scale as follows: 1+ = <25,000 units; 2 + = 25,001-50,000 units; 3+ = 50,001-100,000 units; 4 + = >100,001 units. t H N D = hybridization not detected, m R N A specific for the following clones could not be detected in any of the samples: H-ras, los, p53,

N-myc raf N-ras

IL2 receptor§

abl

bas

sis erb B myc mos myb

pBR322~t

fes fps

Actin Transferrin receptor o~-tubulin K-ras o~-interferon ~interferon

Control

ACCUMULATION IN NON-T-CELL DISORDERS*

No. 12 No. 13 No. 14 No. 15 No. 16 No. 17 No. 18 No. 19 Hairy cell B-cell EBV+ B-cell B-cell B-cell B-cell N o n - H o d g k i n ' s leukemia lymphoma B-lymphoma lymphoma CLL CLL CLL B-lymphoma

TABLE 5. RELATIVE m R N A

O © .v >

Oncogenes and T-lymphoproliferativedisorders cells containing the abnormalities of chromosome 14 which have been reported to occur in some T-cell malignancies and involve genes of the T-cell receptor c~ locus [32, 33]. This is not surprising since the chromosome 14 translocations and inversions have been found in only a low percentage of total T-cell leukemias and lymphomas examined cytogenetically [34]. In addition, in two of these patients the molecular studies were performed on tissues different from those studied for chromosome abnormalities. We examined D N A for amplification of several genes (c-myc, c-[ps, K-ras) in some of these patients (Nos 1-11), but we were unable to identify any gene copy number greater than the control cells [29]. Many reports have detailed activation of protooncogenes, especially members of the ras family, by point mutations [31, 35]. Recent studies by Hirai et al. [36] have used the NIH 3T3 cell transfection assay to detect activated N-ras oncogenes in human leukemic cells. We have not yet tested D N A derived from cells of the T-helper lymphoproliferative disorders for activation of K-ras sequences, though this is a likely mechanism since this gene is elevated in expression in four of six T-helper disorders described here and since K-ras has been shown to be activated in a high percentage of malignancies studied to date [37, 38]. The fps oncogene was originally associated with the feline sarcoma virus and is expressed at high levels in normal bone marrow and myeloid cells [39, 40]. The gene product is a tyrosine kinase that is associated with the cell membrane and/or cytoplasm [41]. It is surprising to find expression of this gene at elevated levels in T-helper lymphoproliferative disorders since expression of this gene has not previously been associated with T-cells. This may reflect an event important in transformation, perhaps via deregulation of a normally non-expressed gene or enhanced expression of a cellular transcript present at low levels in the cell. The results depicted in Table 5 outlining oncogene expression in non-T-cell disorders are consistent with previous reports by other groups. Slamon et aL [11] have also demonstrated expression of myc, myb, Kras and several other genes in a single patient with Hodgkin's disease. In other studies, Roy-Buman et al. have reported the expression ofc-erb B in a variety of lymphoma and leukemia cells [10], including hairy cell leukemia and B-cell lymphoma. From these reports and those of others examining oncogene expression in hematologic malignancies [6-8], it is clear that there is much variation among individuals with the same histologic tumor. However, in many cases a consistent pattern of expression of one or a few genes has emerged, perhaps suggesting some

335

functional significance or some relationship to the mechanism(s) of cellular transformation. Future studies may suggest that tumors should be typed by oncogene involvement and molecular parameters in addition to classical histologic means in an effort to understand mechanisms underlying oncogenesis and to improve predictions of prognosis and possibly treatment. Enhanced expression of transferrin receptor and actin have been associated with both cellular transformation and increased cellular proliferative capacity [42, 43]. The fact that these genes are highly expressed in T-helper and not in T-suppressor disorders may be reflective of the enhanced proliferation and aggressiveness of diseases associated with Thelper malignancies [11]. Under these assay conditions, transferrin receptor and actin were not detectably enhanced in expression upon PHA activation of control PBL. However, longer exposures of the blots did reveal slight but significant increases in m R N A for transferrin receptor and actin in PHAactivated cells. It is, therefore, likely that this enhanced accumulation of mRNA specific for transferrin receptor and actin is caused by the high proliferative capacity of T-helper disorders. Clearly in each of these disorders the percentage of transformed cells in each population varies, making absolute comparisons of the number of copies of specific mRNAs per cell impossible. While transferrin receptor, actin and a~-tubulin transcripts are expressed in normal cycling T-cells, K-ras and fps have not been detected in untransformed lymphocytes or lymphocyte subpopulations. Therefore, it is likely that these events are markers of cellular transformation and/or abnormal cell growth patterns. Indeed, the fact that all of these patients had clonally derived lymphoproliferative disorders has not been established. However, in the few cases where later blood samples could be tested for Tcell receptor t-chain rearrangement, evidence for clonality was found in all those examined (patients Nos 3, 8 and 9; Lust, Banks and Woloschak, unpublished observations). The significance of elevated oncogene expression is unclear. It may be secondary to some primary process responsible for the onset of disease such as increased rates of R N A or DNA synthesis or enhanced cell volume. On the other hand, it is possible that this elevated proto-oncogene expression may be reflective of abnormal genetic control of specific genes known to be important in the regulation of cell growth, perhaps via some transacting activators of transcription or some as yet unidentified mechanisms. It is not yet clearly established that the elevation of expression of normal cellular proto-

336

G.E. WOLOSCHAKet al.

oncogenes in the absence of other aberrancies is sufficient to induce cellular transformation. Acknowledgements--We gratefully acknowledge the support of American Cancer Society Grant No.IM-348, Fraternal Order of Eagles Grant No. 50, a gift from Mrs Edythe Warner and the Mayo Foundation. The authors wish to thank Kathy Jensen for typing this manuscript and Drs Rebecca S. Bahn, Greg Wiseman, and John Lust for helpful suggestions after reviewing the manuscript. This work was presented in part at the 1986 FASEB meetings in St Louis, MO.

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