Immunoreactivity and Receptor Expression of Insulinlike Growth Factor I and Insulin in Human Adrenal Tumors
An Immunohistochemical Study of 94 Cases
Takihiro Kamio, Kazuto Shigematsu, Kioko Kawai, and Hideo Tsuchiyama
From the Second Department of Pathology, Nagasaki University School of Medicine, Nagasaki, Japan
Using immunoperoxidase methods, 94 human ad- renal tumors were examined for evidence of im- munoreactivity and receptor expression of insulin- like growth factor I (IGF-I) and insulin. The fre- quency of IGF-I in adrenocortical carcinomas was significantly higher than that in adenomas of the adrenal glands. The adrenocortical carcinomas showed strong intensity of staining for IGF-I, IGF- I receptors, and insulin receptors. A significant cor- relation between immunoreactivity and receptor expression of both IGF-I and insulin was found only in the adrenocortical carcinomas. The adrenocor- tical adenomas with Cushing’s syndrome and pheochromocytomas, more than adrenocortical adenomas with Conn’s syndrome, also stained strongly for insulin receptors. Thus the IGF-I and insulin probably play a role in the growth of ad- renocortical carcinoma tissues, possibly through autocrine mechanisms. The expression of insulin receptors in adrenocortical adenomas in the pres- ence of Cushing’s syndrome and pheochromocy- tomas may be associated with functions. (Am J Pa- thol 1991, 138:83-91)
Growth factors seem to be involved in the transformation or proliferation of neoplastic cells.1-6 Insulinlike growth factors (IGFs) are homologous to the amino acid se- quences of proinsulin.7,8 Receptors of insulinlike growth factor I (IGF-I), one form of IGFs, are structually similar to insulin receptors.9,10 In addition, each peptide can bind to its own receptor and, to a lesser extent, to the other re- ceptor.9,10 The production of IGF-I-like peptides and the enhancement of IGF-I receptors were noted in human neoplastic cells.2,5,6,11-13
Many factors have been proposed to stimulate adre- nocortical cell proliferation.14 Insulinlike growth factor I and insulin also regulate growth of the adrenal gland as well as maintain specific adrenal cell functions.15-17 Insulinlike growth factor I immunoreactivity is detectable in the ad- renal gland.16-18 In addition, the existence of distinct re- ceptors for both peptides has been reported.16,17,19,20 Little is known of the activity of IGF-I and insulin in adrenocortical carcinomas and adenomas. In rat and human pheochro- mocytoma cells, insulin induces a differentiation or in- crease in the secretion of catecholamines.21-23 Most re- cently, we obtained evidence that epidermal growth factor receptors (EGFr) with the intracellular tyrosin kinase domain24 are expressed in adrenocortical carcinomas, at a high frequency.25 Like EGFr, IGF-I and insulin receptors also display a tyrosin kinase activity,9,10.26,27 followed by RNA synthesis and gene expression.28 Furthermore EGF may synergize with IGF-I to promote the growth of BALB/ c-3T3 cells, a cell line of mouse fibroblast.29 Hence the presence of IGF-I and insulin and their receptors must be investigated to elucidate mechanisms of cell growth and steroidogenesis in adrenal tumors. In the present study, we used an immunohistochemical method to determine whether or not there is receptor expression as well as immunoreactivity for IGF-I and insulin in adrenal tumors, including adrenocortical carcinomas obtained at autopsy, and adrenocortical adenomas and pheochromocytomas obtained during surgery.
Materials and Methods Tissue Samples
Ninety-eight cases of adrenocortical carcinomas listed in the annual report of Pathological Autopsy Cases in Japan
Supported in part by a grant from the Graduates’ Association of Nagasaki University School of Medicine (to T. Kamio, 1989) and a Grant-in-Aid for Scientific Research from the Ministry of Japan (to K. Shigematsu, 1988 and 1989).
Accepted for publication August 28, 1990.
Address reprint requests to Kazuto Shigematsu, MD, PHD, the Second Department of Pathology, Nagasaki University School of Medicine, 12-4 Sakamoto-machi, Nagasaki 852, Japan.
between 1965 and 1982 were collected. To avoid overt autolysis, great deterioration of immunoreactivity or re- ceptor protein after death, paraffin blocks of 64 tissues obtained at autopsy within 3 hours after death13,20,30 were used for the immunohistochemical study. Twenty-three cases of adrenocortical adenomas (nine of Cushing’s syndrome, 11 of Conn’s syndrome, and three nonfunc- tioning) and seven pheochromocytomas were selected from 89 adrenal tumors filed in the Second Department of Pathology, Nagasaki University School of Medicine. The adrenal tumors were diagnosed according to the rules of the World Health Organization31 and the Armed Forces Institute of Pathology.32 Furthermore, the adrenocortical carcinomas examined were histologically graded on the basis of how closely the cells resembled normal cortical cells; G1, well-differentiated type; G2, moderately differ- entiated type; and G3, poorly differentiated type.25,33
Immunohistochemistry
Serial 5-u-thick sections taken from all the paraffin blocks were stained for immunoreactivity and receptors of IGF-I
and insulin. Sections adjacent to the related tissue sections used for the immunostaining were stained with hematoxylin and eosin (H&E). After treatment with normal goat or horse serum for 60 minutes, sections were incubated with the primary antibody at the indicated dilutions: rabbit anti-hu- man polyclonal IGF-I serum 1/100 (amino acids 1 to 70, IGF -; KabiGen AB, Stockholm, Sweden), rabbit anti-human IGF-I receptor serum 1/50 (Upstate Biotechnology Inc, Lake Placid, NY), mouse anti-porcine monoclonal insulin serum 1/200 (Sekisui Biochemical Co., Osaka, Japan), and mouse anti-human monoclonal insulin receptor serum 1/50 (Cosmo Bio Co., Tokyo, Japan). The sections were incubated for 48 hours at 4℃ for IGF-I receptors, for 60 minutes at room temperature for IGF-I and insulin recep- tors, and overnight at 4℃ for insulin. After incubation, the tissue sections were washed three times (30 minutes each) with phosphate-buffered saline (PBS), pH 7.4. Lo- calization of immunostaining was demonstrated by the avidin-biotin peroxidase complex method, using Vectas- tain ABC kits (Vector Laboratories, Burlingame, CA). Par- affin blocks of human squamous cell carcinoma of lung, placental, and pancreatic tissues obtained at surgery were used as positive controls. 13,34,35 For the negative controls, tissues were preincubated with the IGF-I antibody with an
a
b
C
d
excess of unlabeled IGF-I (Peninsula Laboratories), and by applying an irrelevant rabbit or mouse immunoglobulin serum instead of the IGF-I receptor, insulin, and insulin receptor antibodies. Several cases of adrenocortical ad- enomas were examined for a comparison of immunocy- tochemical staining in paraffin and fresh frozen sections.
Data Analysis
The data obtained were expressed as scores ranging from 0 to 1 (1% to 10% positive cells), to 2 (10% to 50% positive cells), to 3 (50% to 100% positive cells), according to the ratio of positive cells in the tumors.25,36 Differences were analyzed by Chi-square test and Kendall’s rank correlation test (Kendall’s tau).
Results
Human squamous cell carcinoma tissues were used for positive controls of IGF-I, whereas human placental tissues for receptors of IGF-I and insulin and human pancreatic tissues for insulin were examined. Insulinlike growth factor I immunostaining was observed in the carcinoma cells (Figure 1a), columnar epithelium, cartilage, and pulmonary vessels. Immunostaining for receptors of IGF-I and insulin was evident in the placental chorionic villi (Figure 1b, c). Islet cells of the pancreas contained insulin immunoreac- tivity (Figure 1d).
With regard to assessments of tissue preparations, a comparison was made of immunocytochemical staining in paraffin and fresh frozen sections. Although the im- munohistochemical procedure used for the paraffin sec- tions provided less intense peroxidase reactions than seen in the fresh frozen sections, there was no significant dif- ference in the ratio of positive cells between both these preparations, when the tissue sections were incubated under the conditions described in the Materials and Meth- ods. There was no evidence of positive immunostaining in the negative control sections.
According to the differentiation, the adrenocortical car- cinomas were classified into the G1 group (16 cases), G2 group (33 cases), and G3 group (15 cases) (Figure 2). As shown in Table 1, 16 of the 64 patients had functioning adrenocortical carcinomas: 13 with Cushing’s syndrome (seven cases; plus virilization, six cases; unknown symp- tom of virilism), one with Conn’s syndrome, and two with adrenogenital syndrome. Twenty-nine were clinically non- functioning and in the remaining 19, the function or lack of it in adrenocortical carcinomas was unknown. The ad- renocortical adenomas examined never contained necro- sis, mitosis, or vascular or capsular invasion.
a
b
C
In the non-neoplastic adrenal gland, strong immuno- staining for IGF-I was observed in the zona reticularis, stained cells were scattered among adrenal medulla tis- sues, and the immunostaining in zona glomerulosa and zona fasciculata cells was minimal. The IGF-I receptors were prominantly localized in the zona reticularis and zona glomerulosa; the medulla demonstrated minimal staining. Conversely the binding of insulin receptor antiserum showed a prominent labeling of the inner zona fasiculata to the zona reticularis cells; the zona glomerulosa, outer zona fasciculata, and medulla showed slightly less im- munoreactivity. The distribution of insulin was almost same
| Histologic types | No. of cases | Positivity (%) | |||
|---|---|---|---|---|---|
| IGF-I | IGF-I receptor | Insulin | Insulin receptor | ||
| Adrenocortical Carcinoma | 64 | 58 (90.6) | 60 (93.8) | 55 (85.9) | 58 (90.6) |
| Cushing's syndrome | 13 | 11 | 12 | 8 | 13 |
| Conn's syndrome | 1 | 1 | 1 | 1 | 1 |
| Adrenogenital syndrome | 2 | 2 | 2 | 2 | 2 |
| Nonfunctioning | 29 | 27 | 26 | 26 | 25 |
| Unknown | 19 | 17 | 19 | 18 | 17 |
| Adrenocortical Adenoma | 23 | 11 (47.8)* | 22 (95.7) | 16 (69.6) | 23 (100) |
| Cushing's syndrome | 9 | 5 | 9 | 7 | 9 |
| Conn's syndrome | 11 | 5 | 10 | 7 | 11 |
| Nonfunctioning | 3 | 1 | 3 | 2 | 3 |
| Pheochromocytoma | 7 | 4 (57.1)+ | 5 (71.4) | 5 (71.4) | 7 (100) |
* x2 = 11.898 (carcinomas vs. adenomas); P < 0.01.
t x2 = 16.369 (carcinomas vs. pheochromocytomas); P < 0.01.
as that of insulin receptors, but the zona glomerulosa also contained a moderate immunoreactivity.
The immunoreactivity of IGF-I and insulin took the form of a fine granular pattern in the cytoplasm of the tumor cells (Figures 3a, 4a). The immunoreaction for receptors of IGF-I and insulin was limited to the cytoplasm (Figures 3b, 4b) and cell membrane (Figures 3c, 4c).
The results of staining for IGF-I, IGF-I receptors, insulin, and insulin receptors in 94 adrenal tumors are summarized in Table 1. Of the 64 cases of adrenocortical carcinomas, 58 (90.6%) showed IGF-I immunoreactivity. The frequency of IGF-I immunostaining in the adrenocortical carcinomas was significantly higher than in the adrenocortical ade- nomas (11 of 23 cases, 47.8%) and pheochromocytomas
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b
C
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d
(four of seven cases, 57.1%) (P < 0.01). There was no difference in the frequency of staining for IGF-I receptors, as expressed in 93.8% of adrenocortical carcinomas, 95.7% of adrenocortical adenomas, and 71.4% of pheo- chromocytomas. The incidences of insulin in adrenocor- tical carcinomas, adrenocortical adenomas, and pheo- chromocytomas were 85.9%, 69.6% and 71.4%, respec- tively, whereas the insulin receptors were expressed in 90.6%, 100%, and 100%, respectively. The frequency of insulin immunoreactivity and insulin receptor expression among these tumors was much the same.
The intensity of staining was examined in the adrenal tumors. The IGF-I, IGF-I receptors, and insulin receptors were strongly stained in the adrenocortical carcinomas more than in the adrenocortical adenomas (Tables 2 and 3). In addition, the adrenocortical adenomas with Cush- ing’s syndrome and pheochromocytomas showed a stronger intensity for insulin receptors than did the adre- nocortical adenomas with Conn’s syndrome (Table 4), although the intensity of IGF-I, IGF-I receptors, and insulin immunostaining apparently was not related to function of the tumor tissues (data not shown).
To determine whether or not the receptor expression correlated with the immunoreactivity level, data were an-
alyzed by Kendall’s tau. No relation was found in the ad- renocortical adenomas and pheochromocytomas (data not shown). Conversely, in the adrenocortical carcinomas, the receptors for IGF-I and insulin significantly correlated with the intensity of IGF-I and insulin immunostaining, re- spectively, the relationship being positive (IGF-I: Kendall’s tau = 0.403, P = 0.0023, insulin: Kendall’s tau = 0.347, P = 0.0080) (Table 5). There was no significant correlation between IGF-I receptors and insulin receptors in the ad- renocortical carcinomas (Table 6).
The relationship between the intensity of IGF-I receptor staining and the histologic grading of adrenocortical car- cinomas was negative (Kendall’s tau = - 0.447, P = 0.004) (Table 7). There was no significant relationship between the histologic grading of the adrenocortical car- cinomas and the intensity of staining for IGF-I, insulin, or insulin receptors (data not shown).
Discussion
We confirmed the presence of IGF-I and insulin immu- noreactivities and receptor expressions in human adrenal tumors, including adrenocortical carcinomas, adrenocor-
| Staining intensity of IGF-I | ||||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| Adrenocortical carcinoma | 6 | 12 | 25 | 21 |
| Adrenocortical adenoma * | 12 | 6 | 4 | 1 |
| Pheochromocytoma | 3 | 1 | 2 | 1 |
* x2 = 23.225 (carcinomas vs. adenomas); P < 0.01.
| Staining intensity of IGF-I receptors | ||||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| Adrenocortical carcinoma | 4 | 20 | 27 | 13 |
| Adrenocortical adenomat | 1 | 13 | 7 | 2 |
| Pheochromocytoma | 2 | 1 | 3 | 1 |
t x2 = 8.786 (carcinomas vs. adenomas); P < 0.05.
tical adenomas, and pheochromocytomas. In the positive control tissues, the distribution of immunostaining was the same as reported. 13,34,35 We used only formalin-fixed and paraffin-embedded tissues. In the initial experiment, paraffin treatment disclosed a lesser sensitivity of the im- munohistochemical assay than was seen in the fresh fro- zen sections. However the ratio of positive cells in both tissue preparations was similar. Moreover there was no evidence of positive staining when the primary antibody was preincubated with an excess of immunogen peptide or when an irrelevant immunogloblin serum was applied.
Immunostaining for both IGF-I and insulin receptors was localized in the cytoplasm, with a fine granular pattern,
| Staining intensity of insulin | ||||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| Adrenocortical carcinoma | 9 | 19 | 19 | 17 |
| Adrenocortical | 7 | 7 | 7 | |
| adenoma | 2 | |||
| Pheochromocytoma | 2 | 1 | 1 | 3 |
No statistical significance.
| Staining intensity of insulin receptors | ||||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| Adrenocortical carcinoma | 6 | 5 | 28 | 25 |
| Adrenocortical adenoma * | 0 | 8 | 9 | 6 |
| Pheochromocytoma | 0 | 1 | 2 | 4 |
* x2 = 11.277 (carcinomas vs. adenomas); P < 0.05.
| Staining intensity | ||||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| Cushing's syndrome * | 0 | 0 | 3 | 6 |
| Conn's syndrome *t | 0 | 5 | 6 | 0 |
| Pheochromocytomat | 0 | 1 | 2 | 4 |
* x2 = 11.919 (Cushing’s syndrome vs. Conn’s syndrome); P < 0.01. t x2 = 8.1820 (Pheochromocytoma vs. Conn’s syndrome); P < 0.05.
as well as in the cell membrane. Receptor-mediated en- docytosis has been described for various ligands, includ- ing IGF-I and insulin.9,37-39 The pathways that the receptor- ligand complexes follow inside a cell can be divided into four major classes,39 and all classes of receptor show the same initial step; the receptor-ligand complexes after binding at the cell surface internalize through coated pits and vesicles, and ultimately enter acidic endosomal com- partments.39 Hence, immunostaining in the cytoplasm is considered to identify the internalized ligand-binding re- ceptor proteins or degraded receptor proteins. This phe- nomenon may be noted especially in the use of paraffin sections.25,34
In the non-neoplastic adrenal glands, there was a similar distribution of immunoreactivity and receptors between IGF-I and insulin, although there were different intensities of staining in each zone of the adrenal gland. Both IGF-I and insulin are involved in the regulation of cell growth and steroidogenesis in the adrenal cortex, in the presence or absence of angiotensin Il and adrenocorticotropic hor- mone (ACTH).16,17 In the adrenal medulla, both peptides also can increase catecholamine secretion from chromaffin cells in response to high K+, with a different magnitude.40 The existence of distinct receptors for IGF-I and insulin in the adrenal glands has been well documented. 16,17,19,20
| IGF-I immunoreactivity | |||||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | ||
| 0 | 2 | 1 | 1 | 0 | |
| IGF-I | 1 | 0 | 7 | 9 | 4 |
| Receptors | 2 | 3 | 3 | 13 | 8 |
| 3 | 1 | 1 | 2 | 9 | |
Kendall’s tau = 0.403; P = 0.0023.
| Insulin immunoreactivity | |||||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | ||
| 0 | 3 | 2 | 1 | 0 | |
| Insulin | 1 | 0 | 4 | 1 | 0 |
| Receptors | 2 | 3 | 9 | 8 | 8 |
| 3 | 3 | 4 | 9 | 9 | |
Kendall’s tau = 0.347; P = 0.008.
Of the adrenocortical carcinomas investigated, 90.6% showed a strong positivity for IGF-I, and the frequency was significantly higher than that seen in the adrenocortical adenomas and pheochromocytomas. The strong intensity of IGF-I receptors also was noted in adrenocortical car- cinomas, although there was no difference in the fre- quency among the adrenal tumors examined. We found no significant relationship between immunoreactivity or receptor expression of IGF-I and function of the tumor tissues. Gourmelen et al41 reported that serum concen- trations of IGF-I for patients with Cushing’s syndrome did not differ from normal values and did not reflect the severity of the hypercortisolism. Insulinlike growth factor I was seen to act as the growth factor for pheochromocytoma cells.22,23 Furthermore, the intensity of IGF-I immunoreac- tivity was positively correlated with that of IGF-I receptors. Adrenocortical carcinomas seem to have characteristics of fetal adrenal glands,33 which express twice the con- centration of IGF-I messenger ribonucleic acid (mRNA) than does the liver.42 Taken in conjunction with the ob- servations that human neoplastic cells produce or release IGF-I-like peptides and express a high level of IGF-I re- ceptors for proliferation of tumor cells,2,5,6,11-13 the possi- bility that IGF-I in particular is associated with growth of adrenocortical carcinomas, possibly through an autocrine mechanism, would have to be considered.
Fitzpatrick et al43 reported that the expression of growth factor receptors significantly correlated with the histologic grading. With regard to assessments of histologic grading of adrenocortical carcinomas, we found negative corre- lation with the intensity of IGF-I receptor staining, thereby indicating that IGF-I receptors in the well-differentiated type were expressed at higher levels than in the poorly differ- entiated type. This finding supports the concept that IGF- I or its receptor is related to the function of differentiation of cells and tissues as well as to growth.44
We found no significantly different frequency of insulin immunoreactivity and insulin receptor expression between benign and malignant tumors of the adrenal glands. Eighty- eight of 94 adrenal tumors examined expressed insulin receptors with different intensities, and only six of the ad- renocortical carcinomas were devoid of positive cells. However the intensity of insulin receptor staining in the
| IGF-I receptors | |||||
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | ||
| 0 | 1 | 1 | 4 | 0 | |
| Insulin | 1 | 1 | 1 | 1 | 2 |
| Receptors | 2 | 2 | 11 | 11 | 4 |
| 3 | 0 | 7 | 11 | 7 | |
Kendall’s tau = 0.210; P = 0.1483.
| Staining intensity of IGF-I receptor | ||||
|---|---|---|---|---|
| 0 | 1 | 2 | 3 | |
| G1 | 1 | 5 | 9 | 1 |
| G2 | 2 | 10 | 12 | 9 |
| G3 | 1 | 5 | 6 | 3 |
Kendall’s tau = - 0.447; P = 0.004.
adrenocortical carcinomas was stronger than that in the adrenocortical adenomas. Like IGF-I receptors, the inten- sity between immunoreactivity and receptor expression of insulin showed a positive correlation in the adrenocor- tical carcinomas. Hence insulin also may play a role in growth of adrenocortical carcinomas, although insulin may be much less potent than IGF-I for the growth.
We obtained no clear evidence of the coexpression of IGF-I and insulin receptors in the same carcinoma cells. However somatomedin C, which is considered to be identical to IGF-I, possesses a structural and functional homology with insulin.7,8 Receptors for IGF-I and insulin also share similar features with two @-subunits and two ß-subunits linked by disulfide bridges.9,10 Insulinlike growth factor I and insulin each bind to its own receptor and, to a lesser extent, to the other receptor,9,10 and the mitogenic effect of insulin is considered to be mediated, in part, through the IGF-I receptors. 45 Therefore, IGF-I and insulin may affect the adrenocortical carcinomas by receptor in- teraction, as well as through their own receptors.
Immunostaining for the insulin receptors disclosed a stronger intensity in the adrenocortical adenomas with Cushing’s syndrome and pheochromocytomas than in the adrenocortical adenomas with Conn’s syndrome. A high concentration of insulin was noted to enhance in- dependently the activities of steroid hydroxylase, such as 36-hydroxysteroid dehydrogenase/isomerase, and 21- and 118-hydroxylase.16,46 It is not clear whether the con- centration of insulin is elevated in patients with Cushing’s syndrome and in those with a pheochomocytoma. How- ever the enhancement of insulin receptor expression does indicate that insulin, acting on its receptor, may play an important role in the regulation of glucocorticoid and cat- echolamine biosynthesis in the adrenocortical adenomas with Cushing’s syndrome and pheochromocytomas. Tsu- gawa et al21 reported that insulin directly increased cat- echolamine secretion from human pheochromocytoma cells, the results of which differed from findings in normal bovine chromaffin cells.40 Whether insulin stimulates ste- roidogenesis in the adrenocortical adenomas with Cush- ing’s syndrome is the subject of ongoing study.
In conclusion, the immunoreactivity and receptor expression of both IGF-I and insulin were evidenced in 94
adrenal tumors. The IGF-I and insulin probably act as au- tocrine growth factor in the adrenocortical carcinomas. In addition, IGF-I may also play a role in the function of dif- ferentiation of the adrenocortical carcinoma tissues. The possibility that the expression of insulin receptors may be associated with steroid-catecholamine biosynthesis in benign adrenal tumors is worthy of consideration. The expression of growth factors in neoplastic tissues may be related to the prognosis of patients.47,48 We were not able to determine whether the growth factors and their receptors can serve as a prognostic parameter, because 96.9% of the adrenocortical carcinomas had already me- tastasized to other organs and because 78.3% of the patients died within 1 year of the onset of symptoms.
Acknowledgments
The authors thank M. Ohara for critical comments, M. Inomata, Y. Yamashita, and S. Nakanose for technical assistance, and T. Hayashida for photographic services. They also thank Dr. M. Nakano, Department of Surgery, Maizuru Municipal Hospital, Kyoto, Japan, for help with collection of the tissue samples and the histological grading of the adrenocortical carcinomas.
References
1. Hendler FJ, Ozanne BW: Human squamous cell lung cancers express increased epidermal growth factor receptors. J Clin Invest 1984, 74:647-651
2. James R, Bradshaw RA: Polypeptide growth factors. Annu Rev Biochem 1985, 53:259-292
3. Reeve AE, Eccles MR, Wilkins RJ, Bell GI, Millow LJ: Expres- sion of insulin-like growth factor-Il transcripts in Wilms’ tumor. Nature 1985, 317:258-260
4. Sporn MB, Roberts AB: Autocrine growth factors and cancer. Nature 1985, 313:745-747
5. Tricoli JV, Rall LB, Karakousis CP, Herrera L, Petrelli NJ, Bell Gl, Shows TB: Enhanced levels of insulin-like growth factor messenger RNA in human colon carcinoma and liposarcoma. Cancer Res 1986, 46:6169-6173
6. Minuto F, Monte PD, Barreca A, Alama A, Cariola G, Giordano G: Evidence for autocrine mitogenic stimulation by somato- medin/insulin-like growth factor 1 on an established human lung cancer cell line. Cancer Res 1988, 48:3716-3719
7. Rinderknecht E, Humbel RE: The amino acid sequence of human insulin-like growth factor I and its structural homology with proinsulin. J Biol Chem 1978, 253:2769-2776
8. Svoboda ME, Van WYK JJ, Klapper DG, Fellows RE, Grissom FE, Shlueter RJ: Purification of somatomedin-C from human plasma: Chemical and biological properties, partial sequence analysis, and relationship to other somatomedins. Biochem- istry 1980, 19:790-797
9. Kaplan SA: Medical progress: The insulin receptor. J Pediatr 1984, 104:327-336
10. Rechler MM, Nissley SP: The nature and regulation of the receptors for insulin-like growth factors. Annu Rev Physiol 1985, 47:425-442
11. Foekens JA, Portengen H, Janssen M, Klijn JGM: Insulin- like growth factor-1 receptors and insulin-like growth factor- 1-like activity in human primary breast cancer. Cancer 1989, 63:2139-2147
12. Kurihara M, Tokunaga Y, Tsutsumi K, Kawaguchi T, Shige- matsu K, Niwa M, Mori K: Characterization of insulin-like growth factor I and epidermal growth factor receptors in meningioma. J Neurosurg 1989, 71:538-544
13. Shigematsu K, Kataoka Y, Kamio T, Kurihara M, Niwa M, Tsuchiyama H: Partial characterization of insulin-like growth factor I in primary human lung cancers using immunohisto- chemical and receptor autoradiographic techniques. Cancer Res 1990, 50:2481-2484
14. Gill GN, Crivello JF, Hornsby PJ, Simonian MH: Growth, function and development of the adrenal cortex: Insights from cell culture, Growth of Cells in Hormonally Defined Me- dia. Edited by GH Sato, AB Pardee, DA Sirbasku. New York, Cold Spring Harbor Laboratory, 1982, pp 461-482
15. Horiba N, Nomura K, Hizuka N, Takano K, Demura H, Shi- zume K: Effects of IGF-I on proliferation and steroidogenesis of cultured adrenal zona glomerulosa cells. Endocrinol Jpn 1987, 34:611-614
16. Penhoat A, Chatelain PG, Jaillard C, Saez JM: Characteriza- tion of insulin-like growth factor I and insulin receptors on cultured bovine adrenal fasciculata cells. Role of these pep- tides on adrenal cell function. Endocrinology 1988, 122:2518- 2526
17. Penhoat A, Naville D, Jaillard C, Chatelain PG, Saez JM: Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells. J Biol Chem 1989, 264:6858-6862
18. Hansson H-A, Nilsson A, Isgaard J, Billig H, Isaksson O, Skottner A, Andersson IK, Rozell B: Immunohistochemical localization of insulin-like growth factor I in the adult rat. His- tochemistry 1988, 89:403-410
19. Pillion DJ, Arnold P, Yang M, Stockard CR, Grizzle WE: Re- ceptors for insulin and insulin-like growth Factor-I in the human adrenal gland. Biochem Biophys Res Commun 1989, 165: 204-211
20. Shigematsu K, Niwa M, Kurihara M, Yamashita K, Kawai K, Tsuchiyama H: Receptor autoradiographic localization of in- sulin-like growth factor-I (IGF-I) binding sites in human fetal and adult adrenal glands. Life Sci 1989, 45:383-389
21. Tsugawa M, Moriwaki K, Miyagawa J, Gomi M, Fujii H, lida S, Tarui S: Induction of differentiation of human phaeo- chromocytoma cells in culture by epidermal growth factor and insulin. Anticancer Res 1987, 7:1161-1164
22. Dahmer MK, Perlman RL: Insulin and insulin-like growth fac- tors stimulate deoxyribonucleic acid synthesis in PC12 pheochromocytoma cells. Endocrinology 1988, 122:2109- 2113
23. Nielsen FC, Gammeltoft S: Insulin-like growth factors are mi- togens for rat pheochromocytoma PC12 cells. Biochem Bio- phys Res Commun 1988, 154:1018-1023
24. Ushiro H, Cohen S: Identification of phosphotyrosin as a product of epidermal growth factor activated protein kinase
in A-431 cell membranes. J Biol Chem 1980, 225:8263- 8265
25. Kamio T, Shigematsu K, Sou H, Kawai K, Tsuchiyama H: Immunohistochemical expression of epidermal growth factor receptors in human adrenocortical carcinoma. Hum Pathol 1990, 21:277-282
26. Rubin JB, Shia MA, Pilch PF: Stimulation of tyrosin-specific phosphorylation in vitro by insulin-like growth factor I. Nature 1983, 305:438-440
27. Kasuga M, Yamaguchi FY, Blithe DL, Kahn CR: Tyrosin- specific protein kinase activity is associated with the purified insulin receptor. Proc Natl Acad Sci USA 1983, 80:2137- 2141
28. Pastan I, Willingham MC: Endocytosis. edited by Pastan I, Willingham MC. New York, Plenum Press, 1985, pp 1-44
29. Leof EB, Wharton W, Van Wyk JJ, Pledger WJ: Epidermal growth factor (EGF) and somatomedin C regulate G1 pro- gression in competent BALB/c-3T3 cells. Exp Cell Res 1982, 141:107-115
30. Shigematsu K, Niwa M, Shimomura C, Kamio T, Ozaki M, Tsuchiyama H: Different subtypes of atrial natriuretic peptide receptors in human fetal, neonatal and adult adrenal glands. Biomed Res 1990, 11:109-115
31. World Health Organization. Histological Typing of Endocrine Tumors. Edited by ED Williams. Geneva, 1980
32. Page DL, Delellis RA, Hough AJ: Tumors of the Adrenal. Washington, DC, Armed Forces Institute of Pathology, 1986
33. Nakano M: Adrenal cortical carcinoma: A clinicopathological and immunohistochemical study of 91 autopsy cases. Acta Pathol Jpn 1988, 38:163-180
34. Yanaihara C, Mochizuki T, Inoue T, Iguchi K, Toyoshige M, Hoshino M, Iwanaga T, Fujita T, Izumi S, Nakane K, Yanaihara N: Immunohistochemical approach to insulin receptor with use of synthetic peptides. Acta Histochem Cytochem 1987, 20:245-250
35. Nestler JE, Williams T: Modulation of aromatase and P450 cholesterol side-chain cleavage enzyme activities of human placental cytotrophoblasts by insulin and insulin-like growth factor I. Endocrinology 1987, 121:1845-1852
36. Wrba F, Reiner A, Ritzinger E, Holzner JH: Expression of epidermal growth factor receptors (EGFR) on breast carci- nomas in relation to growth fractions, estrogen receptor status and morphological criteria: An immunohistochemical study. Pathol Res Pract 1988, 183:25-29
37. Fehlmann M, Carpentier J-L, Van Obberghen E, Freychet P,
Thamm P, Saunders D, Brandenburg D, Orci L: Internalized insulin receptors are recycled to the cell surface in rat he- patocytes. Proc Natl Acad Sci USA 1982, 79:5921-5925
38. Schalch DS, Sessions CM, Farley AC, Masakawa A, Emler CA, Dills DG: Interaction of insulin-like growth factor I/so- matomedin-C with cultured rat chondrocytes: Receptor binding and internalization. Endocrinology 1986, 118:1590- 1597
39. Shepherd VL: Intracellular pathways and mechanisms of sorting in receptor-mediated endocytosis. Trends Pharmacol Sci 1989, 10:458-462
40. Dahmer M, Perlman R: Bovine chromaffin cells have insulin- like growth factor-l (IGF-I) receptors: IGF-I enhances cate- cholamine secretion. J Neurochem 1988, 51:321-323
41. Gourmelen M, Girard F, Binoux M: Serum somatomedin/ insulin-like growth factor (IGF) and IGF carrier levels in patients with Cushing’s syndrome or receiving glucocorticoid therapy. J Clin Endocrinol Metab 1982, 54:885-892
42. Han VKM, Lund PK, Lee DC, D’Ercole AJ: Expression of somatomedin/insulin-like growth factor messenger ribonu- cleic acids in the human fetus: Identification, characterization, and tissue distribution. J Clin Endocrinol Metab 1988, 66: 422-429
43. Fitzpatrick SL, Brightwell J, Wittliff JL, Barrows GH, Schultz GS: Epidermal growth factor binding by breast tumor biopsies and relationship to estrogen receptor and progestin receptor. Cancer Res 1984, 44:3448-3453
44. Chatelain P, Penhoat A, Perrard-Sapori MH, Jaillard Ch, Naville D, Saez J: Maturation of steroidogenic cells: A target for IGF- I. Acta Paediatr Scand [Suppl] 1988, 347:104-109
45. King GL, Kahn CR, Rechler MM, Nissley SP: Direct dem- onstration of separate receptors for growth and metabolic activities of insulin and multiplication-stimulating activity (an insulin-like growth factor) using antibodies to the insulin re- ceptor. J Clin Invest 1980, 66:130-140
46. Cathiard AM, Crozat A, Durand Ph, Saez JM: In vitro spon- taneous and ACTH-dependent maturation of the steroido- genic pathway of ovine fetal adrenal cells. Endocrinology 1985, 116:585-590
47. Neal D, Marsh C, Bennett MK, Abel PD, Hall RR, Sainsbury JRC, Harris AL: Epidermal-growth-factor receptors in human bladder cancer: Comparison of invasive and superficial tu- mors. Lancet 1985, 16:366-368
48. Sainsbury JRC, Farndon JR, Sherbet GV, Harris AL: Epi- dermal-growth-factor receptors and oestrogen receptors in human breast cancer. Lancet 1985, 16:364-366