Insulin and IGF-I stimulate normal and virally transformed T-lymphocyte cell growth in vitro

BRAIN,

BEHAVIOR,

Insulin

AND

IMMUNITY

6,

377-386 (1992)

and IGF-I Stimulate T-Lymphocyte

MITCHELL

E. GEFFNER,*.’

Normal and Virally Transformed Cell Growth in Vitro

NOELLE

BERSCH,~

AND DAVID

W. GOLDEN

Depnrtments of *Pediatrics and fMedicine. UCLA School of Medicine, Los Angeles. 90024: and $Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer Net+, York. NE\%, York 10021

California Center.

We used normal and HTLV-II-transformed T-lymphocytes as target cells to study clonal proliferative responses to physiologic and supraphysiologic concentrations of insulin and SF-I. Responses of both growth factors were measured in the presence and absence of CXIR-3, an IGF-I receptor-blocking antibody. A biphasic response to insulin was noted in all cell lines with the first peak [78 2 6.67~ (means ? SE) above control] occurring at I .4 or 1.6 nmoliliter and a second peak (84 2 4.9% above control) occurring at 18.0 nmoliliter. Following preincubation with aIR-3, the overall clonal profile in response to insulin was significantly reduced CF(7.56) = (10.4. p i .OOOl] as a result of blunting at high physiologic and supraphysiologic insulin concentrations, i.e., 2 1.6 nmoli liter. As expected, the overall clonal profile in response to IGF-I was blocked by (uIR-3 [F(4,32) = 11.6, p < .OOOl]. These data show that insulin at both physiologic and supraphysiologic concentrations. as well as IGF-I, stimulate virally transformed T-lymphoblast growth. The significant inhibition of growth responses to high concentrations of insulin and to IGF-I by aIR-3 suggests mediation of these effects through the IGF-I receptor. Similar studies were performed using freshly isolated, phytohemagglutinin (PHA)-stimulated T-lymphocytes. The overall response to insulin was significantly reduced compared to the profile of transformed T-lymphoblasts [F(7,70) = 4.9, p = .0002] as a result of blunting at physiologic insulin concentrations < 1.8 nmol/liter. In response to IGF-I. the clonal profile of PHA-stimulated T-lymphocytes was slightly reduced compared to that of virally transformed T-lymphoblasts [F(4.40) = 3.4. p = .0174]. Thus, both insulin and IGF-I receptor-effecter mechanisms are involved in the growth of virally transformed T-lymphoblasts. whereas the IGF-I receptor-effecter mechanism appears to play a more significant role in the growth of normal. mitogen-activated T-lymphocytes. C: 199: Academic Prer\. Inc.

INTRODUCTION

While there is a large body of knowledge regarding signals for T-lymphocyte activation and intracellular requirements for signal transduction (Royer & Reinherz, 1987), relatively little is known about the modulation of T-lymphocyte cell growth by hormones mainly concerned with metabolic regulation, such as the insulin family of hormones, i.e.. insulin, insulin-like growth factor-I (IGF-I), and IGF-II. Insulin and IGF-I are critical growth factors for many cells and their biologic responses often overlap and may proceed by interaction either with their own receptors or, in some cases, with both receptors (Nissley & Rechler, 1984; Flier, Usher, & Moses, 1986; Chaiken, Moses, Usher, & Flier, 1986; Nagajaran & Anderson, 1982). T-lymphocytes are known to possess both insulin (Brown, Ercolani, & Ginsberg, 1983; Helderman, Pietri, & Raskin, 1983; Tapson, BoniSchnetzler, Pilch, Center, & Berman, 1988; Kozak, Haskell, Greenstein, Rechler, Waldmann, & Nissley, 1987) and IGF-I (Tapson et al., 1988; Kozak et al., 1987) ’ To whom correspondence

and reprint requests should be addressed 377 0889-1591192 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction m any form reserved.

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receptors; however, the insulin receptor is not displayed on resting T-cells, but appears during activation by antigen, mitogen, or human T-cell leukemia virus (HTLV) transformation. IGF-I receptors can be found on both resting and activated T-cells (Tapson et al., 1988). The T-lymphocyte IGF-I receptor has been shown to mediate chemotactic responsiveness to both insulin and IGF-I (Tapson et al., 1988 and Berman & Center, 1987) and thymidine incorporation in response to IGF-I (Tapson et al., 1988). We show here that insulin and IGF-I exert a direct, growth-promoting action on both normal, mitogen-activated human T-lymphocytes and virally-transformed human T-lymphoblasts through the insulin or IGF-I receptor depending on the prevailing growth factor concentration. MATERIALS

AND METHODS

All studies were performed with the informed consent of the subjects and/or their parents, and with the approval of the UCLA Human Subject Protection Committee. HTLV-II-Transformed

Human

T-Lymphoblasts

T-lymphoblast cell lines were established from live healthy individuals, including one infant. The methodology for peripheral blood T-lymphocyte transformation by the human retrovirus HTLV-II has been described previously (Chen, McLaughlin, Gasson, Clark, & Golde, 1983; Chen, Quan, & Golde. 1983; Saxon, Stevens, & Golde, 1978). Phytohemagglutinin (PHA)- and interleukin-2 (IL-2)primed low-density peripheral blood mononuclear cells (1 x lo’), obtained by Ficoll-Hypaque density-gradient separation of 2-20 ml of blood, were cocultivated with an equal number of lethally irradiated (12,000 cGy) late-passage MO cells in Iscove’s medium supplemented with 20% fetal bovine serum. The MO T-cell line was derived from the spleen of a patient with a variant of hairy-cell leukemia (Saxon et al.. 1978). A virus-infected immortalized T-cell line is produced in about 3 weeks. The resultant T-cell lines are of mature helper-inducer phenotype and constitutively produce lymphokines (Chen et al., 1983). Cell lines were fed 2 days prior to clonogenic studies to ensure that all experiments were conducted with cells in an exponential growth phase. Fifty thousand transformed T-lymphoblasts/ml were cultured in methylcellulose in microtiter plates containing either recombinant human insulin { 1.3, 1.4, 1.6, and 1.8 nmol/l [the concentration range in which peak responsiveness to physiologic levels of insulin is found (Geffner. Kaplan, Bersch, Lippe, Smith, Nagel, Santulli, Li, & Golde, 1987b; Geffner, Golde, Lippe, Kaplan, Bersch, & Li, 1987a; Geffner, Bersch, Nakamoto, Scott, Johnson, & Golde, 1991) and 8.6, 18.0, and 44.8 nmohliter [the concentration range in which peak responsiveness to supraphysiologic levels of insulin is found (Geffner et al., 1987b; Geffner et al., 1991)]}, biosynthetic IGF-I (0.91, 1.05, 1.18, and 1.31 nmohliter [the concentration range in which peak responsiveness to IGF-I is found (Geffner et al., 1987a,b; 1991)]} or phosphatebuffered saline (PBS). In some studies, the T-lymphoblasts were cultured in the presence of 20% fetal calf serum (FCS) whereas in others serum-free media was used containing 0.1% crystalline bovine serum albumin (BSA; Intergen Co., Purchase, NY) without IL-2. The serum-containing experiments were repeated following 1 h of preincubation with aIR-3 (500 r&ml), a monoclonal antibody directed against the IGF-I receptor [raised in mouse ascites fluid and kindly provided by Dr. Steven Jacobs (Van Wyk, Graves, Casella, & Jacobs, 1985; Kull,

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Jacobs, Su, Svoboda, Van Wyk, & Cuatrecasas, 1983)]. After 7 days, colonies containing a minimum of eight cells were enumerated using an inverted microscope. All experiments were performed in triplicate, with a maximal replicate variability of ~5%. Freshiy

Isolated,

PHA-Stimuluted

Normal

Human

T-Lymphocytes

Seven experiments were conducted using normal T-lymphocytes from four healthy subjects. In brief, low-density peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density-gradient separation of 10 ml of blood. Peripheral blood lymphocytes were incubated for 2 h in T-flasks after which nonadherent cells were collected and exposed to 170 PHA (HA15, Burroughs-Wellcome Co., Research Triangle Park, NC) for 24 h. At the end of this incubation, cells were enumerated and adjusted to a final concentration of 5 x lO’/ml. They were then grown in liquid culture in the presence of 20% FCS and IL-2 for either 1, 3, 3.5, 7, or 14 days. The cells were centrifuged, fed fresh medium with IL-2 2 days prior to study, and then washed and resuspended in serum-free medium containing 0.1% crystalline BSA without IL-2. The cells were then plated in methylcellulose and 1.5% crystalline albumin in Nunc 96-well flat-bottomed microtiter plates (Irvine Scientific, Inc., Santa Ana, CA) with insulin, IGF-I, or PBS added as above. Colonies were counted after 14 days. Data Analysis

Data are expressed as means ? SEM. Statistical comparisons of clonal profiles with and without aIR-3 preincubation and of clonal profile differences between virally transformed T-lymphoblasts and PHA-stimulated T-lymphocytes were made by repeated-measure two-factor analysis of variance (ANOVA) followed by analysis of simple main effects (Keppel, 1973) [CLR ANOVA (Clear Lake Research, Houston, TX)] to determine significance of each individual variable at each level of the other independent variable. Comparisons of baseline colony counts were made by Student’s paired t test. Statistical significance is defined as p < .05. RESULTS HTLV-II-Transformed

Human

T-Lymphoblasts

The clonal responses of T-lymphoblast cell lines in response to insulin and IGF-I are shown in Fig. 1. In Fig. lA, T-lymphoblast cell lines were cultured in the presence of insulin under either serum-containing (n = 5) or serum-free (n = 8) conditions with no significant difference in clonal response profiles. Under both conditions, two peaks were observed (which occurred in each individual case). Under serum-containing conditions, the mean first peak response was 78 +- 6.6% above baseline (defined as 100%) occurring at physiologic concentrations of insulin (1.4 or 1.6 nmol/liter) and the mean second peak response was 84 + 4.9% above baseline occurring at the supraphysiologic insulin concentration of 18.0 nmol/liter. Under serum-free conditions, the mean first peak response was 66 ? 4.3% above baseline occurring at an insulin concentration of either 1.4 or 1.6 nmol/liter and the mean second peak response was 79 + 6.6% above baseline occurring at an insulin concentration of 18.0 nmol/liter. In these experiments, baseline colony counts were similar between studies with (35.3 + 0.9) and without

380

GEFFNER,

-I c

BERSCH,

-

T-lymphoblarls rserum jn=5j

--•---

T-IymphoblaSIs

GOLDE

serum (n&I)

Insulin

--PBS

AND

0 92

(nmol/L)

105

1 16

131

(nmol/L) FIG. 1. Augmentation of basal HTLV-II-transformed T-lymphoblast colony formation in response to insulin (A) and IGF-I (B). The x-axis in A and in Figs. 2A and 3A shows the concentrations of insulin employed (1.3, I .4. 1.6, and 1.8 nmoliliter within the physiologic range and 8.6, 18.0, and 44.8 nmoli liter within the supraphysiologic range). The x-axis in B and in Figs. 2B and 3B shows the concentrations of IGF-I employed (0.92, 1.05. 1.18, and 1.31 nmoliliter). Phosphate-buffered saline (PBS) refers to no added hormone. The y-axis here and in all subsequent figures denotes the percentage of colony augmentation above baseline. No significant differences in clonal response profiles were seen with either growth factor when comparing culture conditions with or without serum. To convert insulin concentrations in nmoliliter to ngiml, multiply by 5.81: to convert IGF-I concentrations in nmoliliter to rig/ml, multiply by 7.65. IGF-I

(35.3 k 2.4) serum [t(19) = 0.16, p = NS]. In Fig. IB. T-lymphoblast cell lines were cultured in the presence of IGF-I under either serum-containing (n = 5) or serum-free (n = 5) conditions with no significant difference in clonal response profiles. In these experiments, baseline colony counts were similar between studies with (43.3 k 1.1) and without (40.8 k 3.0) serum [t(19) = -0.63, p = NS]. Preincubation with oIR-3 significantly reduced the overall profile of clonal responsiveness to insulin [by ANOVA, F(7.56) = 10.4, p < .OOOl] (Fig. 2A). By analysis of simple main effects, the effects of aIR-3 preincubation occurred only at insulin concentrations 2 1.6 nmol/liter; 1.6 nmol/liter [F( 1,8) = 6.1, p = .039]; 1.8 nmol/liter [F(l,S) = 6.3, p = .04]; 8.6 nmol/liter [F(1,8) = 32.5, p < .OOOl];

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OF T-CELL

GROWTH

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200 -

A ” 180e

8oj,;--

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1.05

1.18

I31

IGF-I (nmovl) FIG. 2. Augmentation of basal HTLV-II-transformed T-lymphoblast colony formation in response to insulin (A) and IGF-I (B) with and without preincubation with aIR-3. (A) Note the biphasic stimulatory effect of insulin alone upon T-lymphoblast growth and the nearly complete inhibition by oIR-3 at the three higher concentrations of insulin only. (B) Note the growth-promoting effect of IGF-I upon T-lymphoblast growth (mean peak augmentation at I. 18 nmollliter) and the complete inhibition of growth by pretreatment with aIR-3.

18.0 nmol/liter [F(1,8) = 259, p < .OOOl]; and 44.8 nmol/liter [F(1,8) = 42.9, p < .OOOl]. In these experiments, baseline colony counts differed between studies with (40.3 & 3.9) and without (35.3 -+ 0.9) &R-3 It(19) = -5.5, p < .OOOl]; however, these small differences are not likely to significantly affect clonal responses to insulin. The clonal responses of the same T-lymphoblast cell lines to IGF-I are shown in Fig. 2B. The mean peak response to IGF-I was 75 2 6.2% above baseline occurring at 1.8 nmollliter. As expected, the clonal profile in response to IGF-I was significantly blunted following preincubation with cJR-3 [by ANOVA, F(4,32) = 11.6, p < .OOOl vs. no aIR-31; by analysis of simple main effects, significant differences were seen at IGF-I concentrations of 0.91 nmol/ liter [F(1,8) = 48.6, p < .OOOl]; 1.05 nmol/liter [F(1,8) = 66.3, p < .OOOl]; 1.18 nmol/liter [F(1,8) = 32.2, p < .OOOl]; and 1.31 nmol/liter [F(1,8) = 35.2, p < .OOOl]. In these experiments, baseline colony counts differed between studies

382

GEFFNER,

BERSCH, AND GOLDE

with (54.7 ? 1.I) and without (43.3 + 1.1) cJR-3 [t(19) = -3.6, p < .0019]; however, these small differences are not likely to significantly affect clonal responses to IGF-I. Freshly isolated, PHA-Stimulated

Normal Human T-Lymphocytes

When T-lymphocytes were exposed to PHA for 24 h and maintained for up to 14 days in liquid culture, there were significant clonal profile differences in response to insulin compared to the responses seen with transformed T-lymphoblasts [by ANOVA, F(7,70) = 4.9, p = .0002] (Fig. 3A). Statistical differences (as measured by analysis of simple main effects) were seen, however, only with physiologic insulin concentrations < 1.8 nmol/liter: 1.3 nmol/liter [F( 1,lO) = 11.2, p = .007]; 1.4 nmol/liter [F(l,lO) = 18.5,~ = .002]: and 1.6 nmol/liter [F(l,lO) = 12.0, p = .006]. In contrast, normal clonal responsiveness to supraphysiologic

1

10

100

Insulin (nmolk) 200 B 180 -

-Q-8o [ --80

PBS

T.~m@Dxyirr Ill-R ,,wlwwiyr In-R

I

N

0.92

1.05

1 18

131

IGF-I (nmol/L)

3. Augmentation of basal T-cell colony formation in response to insulin (A) and IGF-I (B). (A) Note reduced phytohemagglutin (PHA)-stimulated T-lymphocyte clonal responsiveness to physiologic insulin concentrations in contrast to responses observed with virally transformed T-lymphoblasts. Clonal responsiveness of PHA-stimulated T-lymphocytes to supraphysiologic insulin concentrations is similar to that seen with virally transformed T-lymphoblasts. (B) Note slightly reduced responsiveness to IGF-I of PHA-stimulated T-lymphocytes compared to virally transformed T-lymphoblasts. FIG.

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insulin concentrations from 8.6 to 44.8 nmol/liter was observed. In these experiments, baseline colony counts (29.6 IT 4.8) were similar to those in studies with T-lymphoblasts (35.3 -+ 0.9) [t(19) = 0.69, p = NS]. The overall clonal response profile to IGF-I was slightly reduced compared to that of virally transformed T-lymphoblasts comparably studied [by ANOVA, F(4,40) = 3.4, p = .0174] (Fig. 3B). Statistical differences (as measured by analysis of simple main effects) were seen, however, only with IGF-I concentrations of 1.05 nmol/liter [F(l,lO) = 5.8,~ = .037] and 1.31 nmol/liter [F(l,lO) = 20.2, p = .OOl]. The mean peak clonal response of PHA-stimulated T-lymphocytes to IGF-I was 48 +- 9.1% above baseline occurring at an IGF-I concentration of 1.18 nmol/liter (vs 75 t 6.2% above baseline for virally transformed T-lymphoblasts also occurring at an IGF-I concentration of 1.18 nmol/liter). In these experiments, baseline colony counts (29.6 -+ 4.9) were similar to those in studies with T-lymphoblasts (43.3 + 1.1) [t(19) = 1.75, p = NS]. DISCUSSION

We have shown that cellular growth of HTLV-II-transformed human T-cell lines is stimulated by physiologic insulin concentrations presumably acting through the insulin receptor, and by supraphysiologic insulin concentrations predominantly acting through the IGF-I receptor, the latter completely inhibited by aIR-3, a monoclonal antibody against the IGF-I receptor. Similarly, virally transformed T-cell growth in this system is stimulated by IGF-I, an action which can also be completely blocked by aIR-3. In contrast, the mean clonal responses of lectin-stimulated, normal T-lymphocytes to physiologic concentrations of insulin, even after 14 days in liquid culture, were significantly blunted compared to those of virally transformed T-lymphoblasts stimulated with a similar concentration range of insulin. Responsiveness of lectin-stimulated T-lymphocytes to supraphysiologic concentrations of insulin, the effect of which is presumably mediated through the IGF-I receptor, was identical to that of virally transformed T-lymphoblasts stimulated with insulin. Lectin-stimulated T-lymphocytes demonstrated a degree of responsiveness to IGF-I slightly reduced compared to that seen with virally transformed T-lymphoblasts. Analagous to our findings, Johnson, Jones, and Kozak (1992), using anti-CD3-activated T-lymphocytes, detected peak IGF-I binding after 4 days in culture and negligible insulin binding throughout an 8-day culture process. In these studies, nanomolar concentrations of IGF-I evoked significant T-cell proliferation in temporal association with expression of the IGF-I receptor. In contrast, Kozak et al. (1987) found, in kinetic studies, expression of IGF-I receptors on PHA-activated T-lymphocytes in parallel to those of insulin. However. examination of their time-course data reveals maximal IGF-I binding after 2 days and maximal insulin binding after 3 days of PHA activation. Stuart, Meehan, Neale, Cintron, and Furlanetto (1991) have recently demonstrated that insulin and IGF-I binding to nonactivated blood mononuclear cells was predominantly to monocytes and B-lymphocytes, with receptors to insulin and IGF-I found on only 2% of T-lymphocytes. The reproducible finding of a biphasic T-lymphoblast clonal response to insulin is possibly explained by a saturation effect to stimulation by physiologic insulin concentrations (> 1.8 nmol/liter) presumably acting through the insulin receptor and resulting in “down-regulation” of the growth response at the higher end of this concentration range. This is followed by the onset of the second peak in

384

GEFFNER,

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response to supraphysiologic insulin concentrations (between 8.w4.8 nmol/liter) mediated through the homologous IGF-I receptor (as determined by blockade of this response by (rIR-3). At the higher end of this insulin concentration range, there is another saturation effect to stimulation by insulin. This biphasic response to insulin occurs whether the culture conditions contain FCS or not and, therefore, is not likely to be an artifact of the background concentrations of either insulin or IGF-I in serum. A similar biphasic response occurs with growth hormone (GH) stimulation of normal T-cell lines presumably working through the GH receptor at low GH concentrations and through one or more homologous lactogenie receptors at high GH concentrations (Geffner, Bersch, Lippe, & Golde, 1990). The sensitivity of insulin receptor induction in T-cells may stem from the degree or duration of activation induced by the conditions of viral transformation of PHA exposure. Although resting human T-cells do not express insulin receptors, T-cells activated either by mitogens, such as PHA or concanavalin A, or following allogeneic stimulation, demonstrate specific insulin binding sites (Brown et al., 1983; Helderman et al., 1983; Tapson et al., 1988). Insulin receptors have also been demonstrated on established T-lymphoblast cell lines (Bhathena, Gazdar, Schechther, Russell, Soehnlen, Gritsman, & Recant, 1982; Cordera, Chimini, Bagnasco, & Gherzi, 1984). Mitogen-activated insulin receptors on T-cells appear to be regulated by pH (Brown et al., 1983), recent circulating insulin concentrations (Helderman et al., 1983), and cell-cycle phase (Wang, Joncourt, Kristensen, & De Week, 1984). As in other tissues, the insulin bound to activated human T-cell membrane receptors is rapidly internalized via receptor-mediated endocytosis (Murphy, Bissacia. Cantor, Berger, & Edelson, 1984). That the same phenomenon was observed with unstimulated T-lymphocytes from a patient with a helper T-cell leukemia suggests that internalization was not an artifact of PHAactivation (Murphy et al., 1984). From a functional standpoint, this binding appears to initiate insulin-induced increases in glucose transport, glucose oxidation, lactate oxidation, and amino-acid transport in activated T-cells (Helderman, 198 1; Boyett & Hofert, 1972; Goldtine, Gardner, & Neville, 1972). In addition, insulin has been shown to be chemotactic for mitogen-stimulated human T-lymphocytes at concentrations known to act through its own receptor (Berman & Center, 1987). With resting lymphocytes, chemotaxis could be induced only by supraphysiologic insulin concentrations known to work via the homologous IGF-I receptor (Berman & Center, 1987). Similar to insulin, IGF-I at nanomolar concentrations binds to activated T-lymphocytes (Tapson et al., 1988). In resting T-cells, IGF-I binding has been demonstrated in one study although there were approximately one-tenth as many IGF-I receptors per cell compared to the number found on mitogen-activated T-cells. However, in another study (Stuart et al., 1991), there was almost no demonstrable IGF-I binding to resting T-lymphocytes. These different results could reflect differences in cell separation and resultant purity of cellular preparations. In contrast, clearcut IGF-I binding to malignant T-cell lines has been shown previously (Lee, Rosenfeld, Hintz, & Smith, 1986). Mitogenic effects of IGF-I have been demonstrated in both resting and activated T-cells using thymidine incorporation as the endpoint (Tapson et al., 1988). In addition, similar concentrations of IGF-I are chemotactic for T-lymphocytes. Thus, while the role of IGF-I and/or its receptor in normal T-cell proliferation and function is unclear,

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stronger evidence exists for a role of IGF-I and/or its receptor in malignant T-cell growth. We previously showed that GH stimulates proliferation of both PHA-stimulated normal and leukemic human T-cells in vitro, an effect originally thought to be mediated by direct action of GH (Mercola, Cline, & Golde, 1981). Our corresponding observations of virally transformed T-lymphoblast responsiveness to concentrations of IGF-I as low as 0.13 nmollliter (Geffner et al., 1987a,b), combined with our recent finding that GH-induced T-lymphoblast colony formation is mediated by local production and secretion of IGF-I (Geffner et al., 1990), suggest a physiologic role for IGF-I in regulation of virally transformed T-cell growth. Since insulin also augments T-lymphoblast proliferation at physiologic concentrations (presumably through its own receptor), it may also subserve an important regulatory role in malignant T-cell growth. With mitogenic stimulation of normal lymphocytes, there is progressive expression of IGF-I and insulin receptors, initially for IGF-I and later for insulin. Following viral induction of malignant T-cell transformation, there is potent T-cell growth in response to both IGF-I and insulin consistent with a role for both growth factors in the regulation of malignant T-cell growth. ACKNOWLEDGMENTS This work was supported in part by U.S. Public Health Service Grants CA 30388 and HL 42107 from the National Institutes of Health.

REFERENCES Berman, J. S., & Center, D. M. (1987). Chemotactic activity of porcine insulin for human T-lymphocytes in vitro. .I. Immunol. 138, 2100-2103. Bhathena, S. J., Gazdar, A. F., Schechther. G. P., Russell, E. K., Soehnlen, F. E., Gritsman. A., & Recant, L. (1982). Expression of glucagon receptors on T- and B-lymphoblasts: Comparison with insulin receptors. Endocrinology 111, 684-692. Boyett, J. D.. & Hofert, J. F. (1972). Stimulatory effect of insulin on glucose metabolism of thymus lymphocytes. Horm. Metab. Res. 4, 163-167. Brown, T. J., Ercolani, L., & Ginsberg, B. H. (1983). Properties and regulation of the T lymphocyte insulin receptor. J. Receptor Res. 3, 481494. Chaiken, R. L., Moses, A. C., Usher, P., & Flier. J. S. (1986). Insulin stimulation of amino-isobutyric acid transport in human skin tibroblasts is mediated through both insulin and type I insulin-like growth factor receptors. J. Ch. Endocrinol. Metab. 63, 1181-l 185. Chen, I. S. Y., McLaughlin. J., Gasson, J. C., Clark, S. C.. & Golde. D. W. (1983). Molecular characterization of genome of a novel human T-cell leukemia virus. Nature 305, 502-505. Chen, I. S. Y., Quan, S. G., & Golde, D. W. (1983). Human T-cell leukemia virus type II transforms normal human lymphocytes. Proc. Nat/. Acad. Sci. U.S.A. 80, 7006-7009. Cordera, R.. Chimini, G.. Bagnasco, M.. & Gherzi. R. (1984). Insulin binding on MOLT 4 cells: Effect of a sulfonylurea. Horm. Res. 20, 246-251. Flier, J. S., Usher, P., & Moses, A. C. (1986). Monoclonal antibody to the type I insulin-like growth factor (IGF-I) receptor blocks IGF-I receptor-mediated DNA synthesis: clarification of the mitogenic mechanisms of IGF-I and insulin in human skin fibroblasts. Proc. Nat/. Acad. Sci. U.S.A. 83, 664-668.

Geffner, M. E., Bersch, N., Lippe. B. M., & Golde, D. W. (1990). T-lymphoblast cell lines from Laron dwarfs augment basal colony formation in response to extremely high concentrations of growth hormone. J. C&n. Endocrine/. Metab. 70, 810-813. Geffner, M. E., Bersch, N., Lippe, B. M., Rosenfeld. R. G.. Hintz, R. L.. & Golde, D. W. (1990). Growth hormone mediates the growth of T-lymphoblast cell lines via locally generated insulin-like growth factor I. J. Clin. Endocrinol. Metab. 71, 464-469. Geffner, M. E., Bersch. N., Nakamoto, J. M.. Scott. M., Johnson, N. B.. & Golde, D. W. (1991).

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