(Received for publication, August 8, 1995; and in revised form, September 28, 1995)
From the
The human insulin-like growth factor II (IGFII) gene has been shown to be imprinted for the promoters P2, P3, and P4 but not for the promoter P1 in liver and chondrocytes. Loss of imprinting of the IGFII gene has been found in a variety of human tumors including rhabdomyosarcoma and lung cancer. In this report, we determined whether loss of imprinting in tumors displays a promoter-specific pattern. We examined allelic expression of all four IGFII promoters in rhabdomyosarcoma, lung cancer, and normal skeletal muscle. We demonstrate that the imprinting of all IGFII promoters is relaxed in rhabdomyosarcoma and lung cancer. These data suggest that loss of imprinting of IGFII gene promoters may be regulated coordinately by a common mechanism in these tumors. Unexpectedly, we also found that P1, in addition to P2, P3, and P4 is monoallelically expressed in three informative adult skeletal muscle tissues. This indicates that imprinting of the IGFII promoter P1 occurs in a tissue-specific manner.
Human insulin-like growth factor II (IGFII), ()a
67-amino acid mitogenic peptide, appears to be involved in normal fetal
growth and development(1, 2) . In addition, it has
been shown that abnormally high levels of IGFII mRNA are expressed in a
number of human malignant tumors, and it has been suggested that IGFII
may act as an autocrine or paracrine growth factor, maintaining and
enhancing tumor growth(3, 4, 5) . An
important role for IGFII in oncogenesis was further supported by the
finding that IGFII acts as a second important signal in SV40 large T
antigen-induced tumorigenesis(6) .
Genomic imprinting, or the differential expression of parental alleles of a gene in somatic cells, is now thought to play a role in human disease and cancer(7) . Prader-Willi syndrome and Angelman syndrome are associated with genomic imprinting on chromosome 15q11-q13(8, 9) . Imprinting has also been proposed to be a potential mechanism for Beckwith Wiedemann syndrome with paternal chromosome 11 isodisomy (10) . It has been shown that the IGFII gene is maternally imprinted in both mice and humans(11, 12, 13) . Altered imprinting of the IGFII gene has been found in a number of tumors where IGFII is believed to play a role in pathogenesis, such as Wilms' tumors, rhabdomyosarcoma (RMS), lung cancer, and leiomyosarcoma(12, 13, 14, 15, 16) . Furthermore, it has recently been shown that in Wilms' tumor, loss of imprinting (LOI) of the IGFII gene is associated with reduced expression of H19 mRNA(17, 18) , which has been suggested to act as a tumor suppressor gene(19) . More recently, an important role for LOI of IGFII in cancer was supported by the evidence that transgenic mice expressing SV40 large T-antigen develop insulinomas and that biallelic expression of IGFII appears to be required for progression from adenoma to carcinoma(20) . These studies suggest that deregulation of IGFII genomic imprinting may play an important role in the development of some tumors.
Tissue-specific imprinting of all three IGFII promoters has been reported in both rats and mice(21, 22) . All three rodent IGFII promoters were found to be expressed from the paternal allele exclusively in all tissues except in the choroid plexus and leptomeninges of the central nervous system. While the human IGFII promoters P2, P3, and P4 have their counterparts in the rodent, the promoter P1 appears to be unique to human(1) . It has also been documented that the IGFII promoter P1 directs expression from both parental alleles while the promoters P2, P3, and P4 transcribe mRNA from only one parental allele in liver and chondrocytes(23, 24) . Relaxation of imprinting of IGFII occurs at high frequency in some human tumors overexpressing IGFII(12, 13, 14, 15, 16) . To explore whether or not this relaxation involved all promoters, we examined the allelic expression status of all four IGFII promoters in RMS, lung cancer, and normal skeletal muscle.
Figure 1:
Structure of the human IGFII gene. A, the numbered boxes indicate the nine exons of the
IGFII gene. The locations of four promoters (P1-P4) are
indicated. The coding regions which encode prepro-IGFII are shown as shaded boxes. Parental alleles were distinguished by digestion
using a common HinfI site and the polymorphic ApaI
site. B, total RNA was reverse-transcribed into cDNA. Each
promoter-specific cDNA transcript was amplified using a common primer
p8 and one of four promoter-specific primers (p1a, p2a, p3a, and p4a). C, 50-fold diluted first round PCR products were amplified by
nested PCR using P end-labeled p9 (asterisks) and
one of four promoter-specific primers (p1b, p2b, p3b, and p4b). HinfI digestion resulted in a 141-bp end-labeled fragment,
which was digested further with ApaI. Transcripts that do not
demonstrate genomic imprinting show two alleles: an ApaI-undigested allele A (141 bp) and an ApaI-digested allele B (108 bp). D, to rule out
partial digestion by ApaI, control template B/B alleles were
amplified using p5 and
P end-labeled p7 (asterisks). This product was added to all enzymatic reactions
and generated a 268-bp end-labeled HinfI fragment and a 235-bp
end-labeled ApaI fragment. E, amplification of IGFII
from total RNA of adult muscle. The nested PCR products were sized on a
1% agarose gel with a DNA molecular weight ladder (lane M).
Amplified transcripts from promoters P1, P2, P3, and P4 show expected
1350, 1306, 1291, and 1266 bp,
respectively.
Figure 2:
Allele usage in the four IGFII promoters
in adult skeletal muscles. A, genomic DNA extracted from adult
skeletal muscles of subjects 1 and 2 was PCR-amplified with primer p5
and P end-labeled primer p7. Labeled PCR products were
purified and digested with HinfI or HinfI + ApaI and analyzed on a 6% polyacrylamide-urea gel and
demonstrate heterozygosity for the ApaI site. B,
total RNA from adult muscle of subject 1 was reverse-transcribed into
cDNA, and nested PCR was performed as described in Fig. 1.
Nested PCR products from four promoters (P1-P4) were digested
with HinfI (H, lanes 1, 3, 5, and 7) or HinfI + ApaI (HA, lanes 2, 4, 6, and 8) in the presence of internal
control template and analyzed on 6% polyacrylamide-urea gel. Lane M shows a
P end-labeled ladder. Note that the internal
control template generates a 268-bp HinfI fragment and a
235-bp HinfI + ApaI fragment to indicate
complete digestion.
Figure 3:
Allele usage in the four IGFII promoters
in cancer. A, RMS of subject 4. B, lung carcinoma of
subject 8. cDNA was transcribed from total RNA of RMS of subject 4 and
lung carcinoma of subject 8 and then amplified by PCR as described in Fig. 1. Nested PCR products from four promoters (P1-P4)
were digested with HinfI (H, lanes 1, 3, 5, and 7) or HinfI+ ApaI (HA, lanes 2, 4, 6, and 8). Completed
digestion is demonstrated by a single 268-bp band (HinfI) or
235-bp band (HinfI + ApaI). Lane M shows a P end-labeled
ladder.
It has been demonstrated that biallelic expression of IGFII in Wilms' tumor was associated with hypermethylation of the H19 promoter region (17, 18) . To determine if Wilms' tumor and RMS share common characteristics, we have studied the methylation status of H19 in RMS tumors as well as in normal skeletal muscle. In contrast with Wilms' tumor, no differences in the methylation pattern between the normal skeletal muscle and the RMS tumors with LOI of IGFII were observed either in the promoter region or in the 3`-region of the H19 gene (Table 1). Therefore, we conclude that biallelic IGFII expression is not linked to H19 hypermethylation in RMS.
Our data demonstrate that silent alleles of all four IGFII
promoters are coordinately activated in RMS, indicating that LOI of
IGFII promoters may be regulated coordinately in this tumor. Our data
also show biallelic expression of all four promoters in lung cancer.
Since IGFII promoters P1-P4 span a 20-kb DNA region, our
data support the hypothesis that imprinting may be regulated in a
regional manner(27) . A recent study has shown that deletion of
the H19 gene region in mice disrupted the imprinting of two other
genes, IGFII and insulin, located over 100 kb upstream, implying an
imprinting control center around the H19 gene(27) . A group of
investigators has also identified an imprinting center within a locus
on chromosome 15 responsible for Prader-Willi syndrome and Angelman
syndrome(28) . Furthermore, it has recently been shown that LOI
of IGFII in Wilms' tumor is linked to increased methylation of
the maternal H19 allele (17) , which is located
200 kb
downstream of the IGFII gene(29) . Since epigenetic alterations
at the H19 locus can regulate neighboring imprinted genes located more
than 100 kb away, it is not surprising to find that LOI involves all
IGFII promoters.
The results reported here document the existence of concordant imprinting of all four IGFII promoters in human adult skeletal muscle. While the IGFII promoter P1 can direct expression from both parental alleles in liver and chondrocytes(23) , all informative adult muscles examined expressed P1-specific IGFII transcripts monoallelically. These data demonstrate that imprinting of the IGFII promoter P1 is tissue-specific. Tissue-specific imprinting has also been found in all three rodent IGFII promoters and the mouse insulin-2 gene(30) . All these genes or their promoters express mRNA biallelically in some specific tissues. On the other hand, a number of tumors also transcribed IGFII from both parental alleles. Although the mechanisms responsible for biallelic expression in normal tissues and in cancer are unknown, the processes involved may be similar. A recent study indicated that allele-specific inactivation occurs early postimplantation(31) . After the establishment of imprinting in the early embryo, a repressed allele of an imprinted gene is reactivated in the precursor cells of specific tissues, and this activation is maintained throughout adult life. To date, no association between LOI and stages of tumor development have been found. This implies that activation of a silenced allele may also occur in the precursor of tumor cells.
In Wilms' tumor, biallelic expression of IGFII was shown to be linked to reduced expression of H19 and increased methylation of H19 promoter region(17, 18) . In the RMS tumors with LOI, we did not observe any H19 methylation differences between normal skeletal muscle tissues and tumor tissues. In addition, overexpression of H19 mRNA was observed by Northern blot analysis in the two RMS tumors with biallelic expression of IGFII, using normal muscle tissue as a control (data not shown). These findings indicate that the mechanisms for LOI of IGFII in RMS may be distinct from those in Wilms' tumor in which the model of enhancer competition between H19 and IGFII is supported. One possible explanation is that LOI of IGFII in RMS may involve IGFII methylation but not H19 methylation. We are currently investigating the methylation pattern of four IGFII promoter regions.
Thus the mechanisms for biallelic expression in normal tissues and cancer as well as the precise molecular mechanisms involved in the process of genomic imprinting remain to be determined, and identification of these mechanisms may have important implications for understanding the biology of these tumors.