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Ribonucleic Acid Expression of the Clustered Imprinted Genes, p57^KIP2, Insulin-Like Growth Factor II, and H19, in Adrenal Tumors and Cultured Adrenal Cells¹

Department of Pathology, University of Helsinki (J.L., A.I.K., P.H., R.V.), FIN-00014 Helsinki; and the Department of Pediatrics, Kuopio University Hospital (R.V.), FIN-70210 Kuopio, Finland

Address all correspondence and requests for reprints to: Dr. Raimo Voutilainen, Department of Pathology, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: Raimo.Voutilainen{at}uku.fi

	Abstract

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The recently cloned cyclin-dependent kinase inhibitor gene p57^KIP2 is genomically imprinted and located on human chromosome 11p15.5. This region contains two other imprinted genes, insulin-like growth factor II (IGF-II) and H19, both of which seem to be implicated in adrenal neoplasms. We analyzed the expression of the putative tumor suppressor p57^KIP2 gene by Northern blotting in normal and hyperplastic adrenals, adrenocortical tumors, and pheochromocytomas. The expression of p57^KIP2 messenger ribonucleic acid (mRNA) correlated positively with H19 and negatively with IGF-II RNA in adrenocortical tissues. p57^KIP2 mRNA (and H19 RNA) was abundantly expressed in normal human adrenals, adrenocortical adenomas from patients with Cushing’s or Conn’s syndrome or without clinical evidence of hormone overproduction, hyperplastic adrenals, and tumor-adjacent adrenal tissues, in which IGF-II mRNA expression was low. In most adrenocortical carcinomas and virilizing adrenal adenomas, very low levels of both p57^KIP2 and H19 RNAs were observed, whereas IGF-II was highly expressed. In pheochromocytomas, p57^KIP2 and H19 RNA expression was highly variable, but on the average it was about 45% and 27%, respectively, of that in normal and tumor-adjacent adrenals.

In cultured adrenocortical cells, ACTH and dibutyryl cAMP treatment slightly reduced the predominant 1.7-kilobase (kb) transcript of p57^KIP2 gene, but induced a 2.5-kb transcript with a simultaneous increase in H19 RNA expression. The stimulatory effect of ACTH on the 2.5-kb p57^KIP2 and H19 transcript accumulation was enhanced by exogenous IGF-II and IGF-I. Our data show that p57^KIP2 and H19 RNAs are expressed usually in parallel in normal and pathological adrenocortical tissues. The decreased expression of both p57^KIP2 and H19 RNAs in conjunction with elevated IGF-II mRNA expression in hormonally active adrenocortical carcinomas suggests that the loss of expression of the putative tumor suppressor genes p57^KIP2 and H19 may be involved in the pathogenesis of these neoplasms.

	Introduction

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INHIBITORS of cyclin-dependent kinases have emerged as potential mediators of cell cycle exit and maintenance of the nonproliferative state. A structurally distinct member of the p21^CIP1 cyclin-dependent kinase family, p57^KIP2, was recently cloned (1, 2). There is a strong correlation between the arrest of cell proliferation and p57^KIP2 expression, and this gene is expressed at high levels mainly in terminally differentiated cells, such as skeletal muscle, heart, kidney, lung, pancreas, and brain, suggesting that expression of p57^KIP2 is coupled to activation of cell differentiation and decision to exit the cell cycle (1, 2). Both human and mouse p57^KIP2 genes were genomically imprinted (with preferential expression from the maternal allele) and mapped to a region within a cluster of imprinted genes, including insulin-like growth factor II (IGF-II) and H19 (3, 4, 5). The putative tumor suppressor gene H19 (6) is also maternally expressed (7, 8), whereas the IGF-II gene is usually paternally active (9, 10, 11, 12). The human p57^KIP2 gene is located on chromosome 11p15.5, a region implicated in distinct adult and childhood neoplasias, including bladder, lung, ovarian, kidney, testicular, hepatocellular, and adrenocortical carcinomas; Wilms’ tumor; and rhabdomyosarcoma (13, 14). The expression pattern, antiproliferative function, chromosomal location, and genomic imprinting make the p57^KIP2 a candidate tumor suppressor gene.

It was recently reported that most Wilms’ tumors express p57^KIP2 and H19 ribonucleic acid (RNA) at reduced levels, associated with increased IGF-II messenger RNA (mRNA) (15). We found previously that the H19 gene is expressed at low levels also in hormonally active adrenocortical carcinomas and pheochromocytomas (16), where IGF-II mRNA is highly expressed (17), a situation similar to the Wilms’ tumors. Therefore, we studied p57^KIP2 RNA expression in normal human adrenals and adrenal neoplasms and compared it with IGF-II and H19 RNA levels to further clarify the significance of these clustered imprinted genes in adrenal pathophysiology. As H19 RNA accumulation is up-regulated by ACTH in human adrenocortical cells (16), we also studied whether a similar regulation of p57^KIP2 mRNA occurs in cultured human adrenal cells.

	Materials and Methods

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Tissues

Normal adrenals were obtained from 5 patients who underwent nephrectomy for kidney tumors. Adrenal medullary tissue was carefully dissected from four normal adrenals. Pathological adrenal glands from 36 patients were obtained during the operations performed at the Department of Surgery, Helsinki University Central Hospital. The tissues investigated included adrenocortical adenomas [from patients with Cushing’s or Conn’s syndromes or without clinical evidence of steroid overproduction (nonfunctional)], virilizing adrenal adenomas, nodular and bilateral adrenocortical hyperplasias, hormonally active adrenocortical carcinomas (from patients with Cushing’s syndrome, hypermineralocorticoidism or virilism), nonfunctional adrenocortical carcinomas (without clinical evidence of steroid overproduction), pheochromocytomas, and adrenal glands adjacent to the tumor tissues (Table 1). Tissue processing and histological diagnostics were described previously (16).

Small pieces of normal and pathological tissues were briefly frozen in liquid nitrogen and then stored at -70 C. The remaining tissues were prepared for primary cultures and treated as described previously (16). The test agents were added as single doses. The growth and morphological characteristics of the cultured cells were assessed by phase contrast light microscopy. All experiments were performed in triplicate wells and repeated at least twice with tissues from different patients.

RNA analysis

Total RNA was isolated from the frozen tissues by ultracentrifugation through a cesium chloride cushion (18). Cytoplasmic RNA was extracted from the cultured cells (19). Northern blotting and hybridizations were performed as previously described (16, 20). The relative intensities of autoradiographic signals were quantitated by densitometric scanning. All data shown here were normalized with the respective 28S RNA values. The correlations between relative levels of different RNAs in in vivo samples were analyzed by Spearman’s test. Differences in RNA levels were assessed by the Mann-Whitney test. The level of significance was chosen as P < 0.05.

Probes

The probe for human p57^KIP2 mRNA was a synthetic oligonucleotide prepared at the Institute of Biotechnology, University of Helsinki. The sequence was 5'-AAG TCG TAA TCC CAG CGG TTC TGG TCC TCG-3', corresponding to the nucleotides 425–454 of the human p57^KIP2 mRNA (GenBank accession no. U22398) (2). The riboprobe for human H19 RNA (8) and complementary DNA probes for human IGF-II (21) and mouse ribosomal 28S RNA (used as a loading control) (22) were labeled as described previously (16).

	Results

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We screened 60 human adrenal samples for p57^KIP2 mRNA expression and detected the highest p57^KIP2 RNA levels in a fetal adrenal (Fig. 1 and Table 1). In most normal adult adrenals or tumor-adjacent adrenal tissues, p57^KIP2 mRNA was readily detectable in Northern blots. The Northern blots hybridized with the p57^KIP2 oligonucleotide probe showed a predominant transcript of approximately 1.7 kilobases (kb) in size in both normal and pathological adrenal tissues ( Figs. 1–4). In some samples, another transcript approximately 1.3 kb in size was detectable. The relative amounts of p57^KIP2 mRNA from all specimens studied are summarized in Table 1, and the most striking changes are shown graphically in Fig. 5. In general, p57^KIP2 RNA expression in normal adult adrenals was approximately 40% of that in fetal adrenals. The p57^KIP2 mRNA levels did not vary significantly among normal adrenals; adrenal medulla; Conn’s, Cushing’s, and clinically nonfunctional adrenocortical adenomas; adrenocortical hyperplasias; and tumor-adjacent adrenal glands. On the other hand, adrenocortical carcinomas from patients with Cushing’s syndrome, hyperaldosteronism, or virilism or from those without clinical evidence of steroid overproduction contained little p57^KIP2 mRNA (P < 0.05 compared with normal adrenals; Figs. 2 and 5 and Table 1). Low p57^KIP2 RNA expression was also detected in virilizing adrenal adenomas (P < 0.05 compared with normal adrenals; Figs. 3 and 5 and Table 1). In pheochromocytomas, p57^KIP2 RNA expression was, on the average, lower than that in normal or tumor-adjacent adrenal tissues, but a considerable variation in p57^KIP2 mRNA abundance was observed (Fig. 4 and Table 1).

View larger version (20K): [in this window] [in a new window]   Figure 1. Comparison of p57KIP2, IGF-II, and H19 RNA expression in fetal adrenal, a virilizing adrenocortical carcinoma, and the tumor-adjacent adrenal. Total RNA was extracted from frozen tissues. The Northern blot was prepared with 20 µg RNA in each lane, and the RNA was transferred onto a nylon membrane. The filter was sequentially hybridized with 32P-labeled p57KIP2, H19, IGF-II, and 28S RNA probe.

View larger version (68K): [in this window] [in a new window]   Figure 2. Comparison of p57KIP2, IGF-II, and H19 RNA expression in normal adult adrenals, functional adrenocortical carcinomas (from a patient with Cushing’s syndrome and a subject with hyperaldosteronism), nonfunctional carcinoma, adrenocortical adenomas, nodular adrenocortical hyperplasia, and adjacent adrenals. The extraction of total RNA, and Northern blot and hybridization conditions were the same as those in Fig. 1.

View larger version (27K): [in this window] [in a new window]   Figure 3. Expression of p57KIP2, IGF-II, and H19 RNAs in normal adrenal, virilizing adrenal adenoma, Cushing’s carcinoma, and bilateral adrenal hyperplasia (left and right adrenals from the same patient). The extraction of total RNA, and Northern blot and hybridization conditions were the same as those in Fig. 1.

View larger version (52K): [in this window] [in a new window]   Figure 4. Expression of p57KIP2, IGF-II, and H19 RNAs in pheochromocytomas and tumor-adjacent adrenal glands. The extraction of total RNA, Northern blot, and hybridization conditions were the same as those in Fig. 1.

View larger version (24K): [in this window] [in a new window]   Figure 5. Relative RNA levels of p57KIP2, H19, and IGF-II in normal adult adrenals, Cushing’s and virilizing adenomas, adrenocortical carcinomas (pooled data from both functional and nonfunctional types), and pheochromocytomas (pheo). The values were calculated as described in Table 1. Means and upper ranges are shown in the figure. The means of the RNA values from normal adrenals were adjusted to 100.

In cultured normal adrenocortical cells, p57^KIP2 mRNA expression was maintained for at least 2 weeks. After 24 h of treatment, ACTH (30 nmol/L) slightly decreased (20–40%; P < 0.01; n = 6) the level of the 1.7-kb p57^KIP2 transcript. This inhibition was dose dependent and detectable from a concentration of 30 pmol/L, with the maximal reduction reached at 3 nmol/L ACTH. In some experiments, there appeared a new transcript (2.5 kb) after 24 h of ACTH stimulation (Fig. 6). Although IGF-II (100 ng/mL) or IGF-I (100 ng/mL) alone had no significant effect on the accumulation of p57^KIP2 mRNA, they augmented ACTH induction of the 2.5-kb transcript under serum-free conditions (P < 0.05). Dibutyryl cAMP [(Bu)₂cAMP; 1 mmol/L] and cholera toxin (15 ng/mL) showed effects on p57^KIP2 mRNA expression similar to those of ACTH (data not shown). In contrast, the protein kinase C activator 12-O-tetradecanoyl phorbol acetate (TPA; 160 nmol/L) and the inhibitor staurosporine (50 nmol/L) had no significant effect on basal or ACTH-modified expression of p57^KIP2 mRNA. The nonspecific protein kinase inhibitor H-7 (100 µmol/L) reduced the expression of the 1.7-kb transcript (P < 0.05), but no induction of the 2.5-kb transcript was detected (Fig. 7). As reported previously (16), H19 gene expression was also induced by ACTH (Fig. 6).

View larger version (53K): [in this window] [in a new window]   Figure 6. The effects of ACTH (30 nmol/L), IGF-II (100 ng/mL), and IGF-I (100 ng/mL) on p57KIP2 and H19 RNA accumulation in primary culture of normal adult adrenal cells. The dispersed cells were allowed to grow for 6 days before the agents were added for further 24 h. Cytoplasmic RNA was extracted, and the Northern blot was prepared with 10 µg RNA in each lane. The experiment was repeated with cells from different patients, and the results were comparable.

View larger version (53K): [in this window] [in a new window]   Figure 7. The effects of H-7 (100 µmol/L), TPA (160 nmol/L), and staurosporine (ST; 50 nmol/L) on p57KIP2 mRNA accumulation in cultured adrenocortical cells. The culture and RNA analysis were the same as those in Fig. 6. The experiment was repeated with cultured cells from three patients, and the results were similar.

View larger version (70K): [in this window] [in a new window]   Figure 8. The effects of (Bu)2cAMP (1 mmol/L), TPA (160 nmol/L), and staurosporine (ST; 50 nmol/L) on p57KIP2 mRNA accumulation in primary culture of pheochromocytoma cells. The dispersed cells were allowed to grow for 7 days before the agents were added for 3 days of treatment. The Northern blot was prepared as described in Fig. 6. The experiment was repeated twice with cells from different patients, and the results were comparable.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our present data show that the p57^KIP2 mRNA is abundantly expressed in normal adrenals, most benign adrenocortical tumors, and hyperplastic adrenals. As in the case of the H19 gene, p57^KIP2 mRNA expression seems to be lower in adult than in fetal adrenals. There are several apparently alternatively spliced p57^KIP2 transcripts in human adrenal tissues, with the predominant one 1.7 kb in size, in agreement with the previous reports (2, 15). Most adrenocortical carcinomas expressed very little p57^KIP2 mRNA. This low p57^KIP2 expression was associated with low H19 and high IGF-II mRNA levels. The inability of the reduced p57^KIP2 and H19 to appropriately halt growth in association with the increased proliferative effect of IGF-II may have promoted cancer development or progression in adrenal tissue. Therefore, the distinct expression pattern of these clustered imprinted genes in adrenal carcinomas suggests that loss of p57^KIP2 expression may have a role in malignant transformation of these neoplasms. The generally parallel expression pattern of p57^KIP2 and H19 RNA in human adrenal tissues raises the possibility that these two genes have a common regulatory pathway. Expression of these two genes is also coordinately reduced in most Wilms’ tumors, and this was suggested to be due to the loss or aberrant imprinting of the maternal alleles (15, 27). However, in our adrenocortical carcinoma samples, the expression of p57^KIP2 and H19 RNAs was not always parallel. In one nonfunctional adrenocortical carcinoma, H19 expression was relatively high, but p57^KIP2 mRNA expression was consistently low in all carcinomas.

Low expression of p57^KIP2 and H19 RNAs in conjunction with high levels of IGF-II mRNA in virilizing adrenal adenomas was unexpected, as other adrenocortical adenomas expressed these RNAs similarly to normal adrenals. This distinct expression pattern of these genes in virilizing adrenal adenomas suggests that the mechanism of this tumor development may be different from that in other adrenal adenomas. Gicquel et al. recently (23) also reported a virilizing adrenal adenoma with high IGF-II mRNA expression.

Responsiveness of p57^KIP2 mRNA expression to the physiological hormone ACTH in parallel with steroidogenesis (20) suggests that p57^KIP2 may play a role in the maintenance of normal adrenocortical differentiation, in agreement with the observation that most p57^KIP2-expressing cells are terminally differentiated (2). In cultured adrenocortical cells, a new transcript appeared after ACTH treatment. This approximately 2.5-kb transcript seems to be an alternatively spliced premature p57^KIP2 RNA, as it appeared in association with reduced predominant 1.7-kb transcript. This suggests that ACTH may regulate the splicing pattern of the premature p57^KIP2 RNA. Alternative splicing of p57^KIP2 RNA was previously described in other human tissues (2, 15, 28). The regulation of the abundance of different p57^KIP2 mRNA transcripts by ACTH is at least in part through the cAMP-dependent protein kinase A pathway, similarly to the regulation of H19 gene expression. The regulation of p57^KIP2 mRNA accumulation seems to be tissue specific. In both adrenocortical and pheochromocytoma cells, p57^KIP2 mRNA expression was regulated through the cAMP-dependent protein kinase pathway. However, (Bu)₂cAMP increased the 1.7-kb transcript of p57^KIP2 gene in pheochromocytoma cells, whereas it reduced it in adrenocortical cells. There may be mechanistic differences in the maintenance of imprinting at p57^KIP2 and H19 loci, as the demethylating drug 5-aza-2'-deoxycytidine increased H19 expression in rhabdomyosarcoma RD cells, but did not activate p57^KIP2 expression (15).

In summary, we observed low expression of p57^KIP2 mRNA in adrenocortical carcinomas and virilizing adrenal adenomas. The ACTH-dependent regulation of adrenocortical p57^KIP2 RNA expression suggests that this gene may have some physiological role in normal adrenocortical growth and/or differentiation. Loss of both p57^KIP2 and H19 expression in conjunction with high IGF-II expression seems to be associated with malignant behavior in hormonally active adrenocortical carcinomas.

	Acknowledgments

 
Ms. Merja Haukka and Ms. Eija Heiliö are thanked for their technical assistance. We appreciate Dr. Anne Ferguson-Smith’s (Cambridge, UK) help in carefully reading and revising the language of the manuscript. Prof. Abraham Hochberg (Jerusalem, Israel) kindly provided the H19 probe.

	Footnotes

 
¹ This work was supported by the Ida Montin Foundation, the Cancer Society of Finland, the Culture Foundation of Finland (to J.L.), the Sigrid Juselius Foundation, and the Nordisk Insulin Foundation (to R.V.).

Received December 11, 1996.

Revised February 11, 1997.

Accepted February 20, 1997.

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