Mannose 6-phosphate/insulin-like Growth Factor 2 Receptor Research Articles

English

The multifunctional transmembrane mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2-R) has been proposed as a putative prognostic marker in breast cancer. Loss of heterozygosity (LOH) with mutations in the remaining allele have been described at the gene level. We previously reported significant decrease of M6P/IGF2-R at the protein level in cancer cells than normal peritumoral ducts of breast tumors which may indicate an increased release of the receptor from the cell membrane to the blood. To assess that hypothesis we measured in this study M6P/IGF2-R levels in serum of women with breast cancer by competitive enzyme-linked immunosorbent assay (ELISA) that allows a detectable sensitivity of 300 ng/mL. The results were obtained referring to a standard curve consisting in varying concentrations of human purified M6P/IGF2-R. Women sera consisted of cancer-free sera (n=39) or serum from women with breast cancer (n=39) were preincubated with anti-M6P/IGF2-R immunoglobulin Y (IgY) antibodies. High levels of M6P/IGF2-R (p<0.01) were found in serum from women with breast cancer in comparison with cancer-free serum. Overall the results support the hypothesis of increased release of the receptor from cancer cells to the blood and add one more argument for M6P/IGF2-R studying as a potential breast cancer marker. Key words: Mannose-6-Phosphate/Insulin-like Growth Factor-2 Receptor (M6P/IGF2-R), loss of heterozygosity (LOH), suppressor gene, breast cancer, competitive enzyme-linked immunosorbent assay (ELISA), IgY, biological marker.

The Structure and Biological Function of CREG.

The cellular repressor of E1A-stimulated genes (CREG) is a 220 amino acid glycoprotein structurally similar to oxidoreductases. However, CREG does not have enzymatic activities because it cannot bind to the cofactor flavin mononucleotide. Although CREG can be secreted, it is mainly an intracellular protein localized in the endocytic-lysosomal compartment. It undergoes proteolytic maturation mediated by lysosomal cysteine proteases. Biochemical studies have demonstrated that CREG interacts with mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R) and exocyst Sec8. CREG inhibits proliferation and induces differentiation and senescence when overexpressed in cultured cells. In Drosophila, RNAi-mediated knockdown of CREG causes developmental lethality at the pupal stage. In mice, global deletion of the CREG1 gene leads to early embryonic death. These findings establish an essential role for CREG in development. CREG1 haploinsufficient and liver-specific knockout mice are susceptible to high fat diet-induced obesity, hepatic steatosis and insulin resistance. The purpose of this review is to provide an overview of what we know about the biochemistry and biology of CREG and to discuss the important questions that remain to be addressed in the future.

Functional evolution of IGF2:IGF2R domain 11 binding generates novel structural interactions and a specific IGF2 antagonist

Among the 15 extracellular domains of the mannose 6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R), domain 11 has evolved a binding site for IGF2 to negatively regulate ligand bioavailability and mammalian growth. Despite the highly evolved structural loops of the IGF2:domain 11 binding site, affinity-enhancing AB loop mutations suggest that binding is modifiable. Here we examine the extent to which IGF2:domain 11 affinity, and its specificity over IGF1, can be enhanced, and we examine the structural basis of the mechanistic and functional consequences. Domain 11 binding loop mutants were selected by yeast surface display combined with high-resolution structure-based predictions, and validated by surface plasmon resonance. We discovered previously unidentified mutations in the ligand-interacting surface binding loops (AB, CD, FG, and HI). Five combined mutations increased rigidity of the AB loop, as confirmed by NMR. When added to three independently identified CD and FG loop mutations that reduced the koff value by twofold, these mutations resulted in an overall selective 100-fold improvement in affinity. The structural basis of the evolved affinity was improved shape complementarity established by interloop (AB-CD) and intraloop (FG-FG) side chain interactions. The high affinity of the combinatorial domain 11 Fc fusion proteins functioned as ligand-soluble antagonists or traps that depleted pathological IGF2 isoforms from serum and abrogated IGF2-dependent signaling in vivo. An evolved and reengineered high-specificity M6P/IGF2R domain 11 binding site for IGF2 may improve therapeutic targeting of the frequent IGF2 gain of function observed in human cancer.

Soluble M6P/IGF2R Released by TACE Controls Angiogenesis via Blocking Plasminogen Activation

The urokinase plasminogen activator (uPA) system is among the most crucial pericellular proteolytic systems associated with the processes of angiogenesis. We previously identified an important regulator of the uPA system in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R). Here, we wanted to clarify whether and how did the soluble form of M6P/IGF2R (sM6P/IGF2R) contribute to modulation of the uPA system. By using specific inhibitors and RNA interference, we show that the tumor necrosis factor α convertase (TACE, ADAM-17) mediates the release of the ectodomain of M6P/IGF2R from human endothelial cells. We demonstrate further that sM6P/IGF2R binds plasminogen (Plg) and thereby prevents Plg from binding to the cell surface and uPA, ultimately inhibiting in this manner Plg activation. Furthermore, peptide 18-36 derived from the Plg-binding site of M6P/IGF2R mimics sM6P/IGF2R in the inhibition of Plg activation and blocks cancer cell invasion in vitro, endothelial cell invasion in vivo, and tumor growth in vivo. The interaction of sM6P/IGF2R with Plg may be an important regulatory mechanism to inhibit migration of cells using the uPA/uPAR system.

Glycosylation-independent binding to extracellular domains 11–13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic modulation of vascular smooth muscle cells

The bio-effects of cellular repressor of E1A-stimulated genes (CREG) have been proposed to depend on its N-glycosylation and binding to mannose-6-phosphate/insulin-like growth factor-2 receptor (M6P/IGF2R). The present study aimed to investigate the detailed mode and specific sites for their binding and the functional relevance of this binding in the phenotypic modulation of vascular smooth muscle cells (SMCs). Wild-type and glycosylation mutant human CREG (wtCREG and mCREG) proteins were expressed and isolated from HEK293 cells. CREG knocked-down SMCs were used to evaluate their biological activity. Both wtCREG and mCREG arrest cell cycle progression of CREG knocked-down SMCs when added to the culture medium. In vitro binding assay revealed that CREG bound to M6P/IGF2R extracellular domains 7–10 and 11–13 in a glycosylation-dependent and -independent manner, respectively. Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody suggest that the binding to domains 11–13, as well as to 7–10, is adequate for CREG to modulate SMC proliferation. These data suggest that soluble CREG protein can exert its biological function via glycosylation-independent binding to the extracellular domains 11–13 of cell surface M6P/IGF2R, and thereby provide novel insights into CREG modulation of SMC phenotypic switching from contractile to proliferative.

Mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) in carcinogenesis

The cation-independent mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is a multifunctional receptor. It is involved in a variety of cellular processes which become dysregulated in cancer. Its tumor suppressor role was recognized a long time ago. However, due to its multifunctionality, it is not easy to understand the extent of its relevance to normal cellular physiology. Accordingly, it is even more difficult understanding its role in carcinogenesis. This review presents critical and focused highlights of data relating to M6P/IGF2R, obtained during more than 25 years of cancer research.

IGF2R polymorphisms and risk of esophageal and gastric adenocarcinomas

The mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a protein that plays a critical role in tumor suppression, in part by modulating bioavailability of a potent mitogen, insulin-like growth factor-2 (IGF2). We tested the hypothesis that the common nonsynonymous genetic variants in M6P/IGF2R c.901C > G (Leu > Val) in exon 6 and c.5002G > A (Gly > Arg) in exon 34 are associated with risk of esophageal and gastric cancers. Study participants in this population-based study comprise 197 controls and 182 cases, including 105 with esophageal-gastric cardia adenocarcinoma (EGA), 57 with noncardia gastric adenocarcinoma and 20 with esophageal squamous (ES) cell carcinoma. Among white males, odds ratios (ORs) were elevated in relation to carrying at least 1 c.901C > G allele for EGA [OR = 1.9; 95% confidence intervals (CIs) = 1.0-3.6] and noncardia gastric cancer (OR = 2.5; 95% CI = 1.2-5.5), but not ES. Exploratory subgroup analyses suggested that associations between EGA and this variant were stronger among irregular or nonusers of nonsteroidal anti-inflammatory drugs (NSAIDs) (OR = 2.3; 95% CI = 1.2-4.2) and cigarette smokers (OR = 2.1; 95% CI = 1.0-4.2). An association between carrying the c.5002G > A genotype and EGA was not evident. These findings suggest that nonsynonymous polymorphisms in M6P/IGF2R may contribute to the risks of EGA and noncardia adenocarcinomas. Larger studies are required to confirm these findings.

Clinical significance of loss of heterozygosity for M6P/IGF2R in patients with primary hepatocellular carcinoma

To investigate the relationship between loss of heterozygosity (LOH) for mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and the outcomes for primary HCC patients treated with partial hepatectomy. The LOH for M6P/IGF2R in primary HCC patients was assessed using six different gene-specific nucleotide polymorphisms. The patients studied were enrolled to undergo partial hepatectomy. M6P/IGF2R was found to be polymorphic in 73.3% (22/30) of the patients, and of these patients, 50.0% (11/22) had tumors showing LOH in M6P/IGF2R. Loss of heterozygosity in M6P/IGF2R was associated with significant reductions in the two year overall survival rate (24.9% vs 65.5%; P=0.04) and the disease-free survival rate (17.8% vs 59.3%; P=0.03). These results show M6P/IGF2R LOH predicts poor clinical outcomes in surgically resected primary HCC patients.

Radiation‐induced posttranscriptional control of M6P/IGF2r expression in breast cancer cell lines

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2r), a member of the IGF axis of growth factors, is a negative regulator of cell growth and a putative tumor suppressor gene. Regulation of M6P/IGF2r levels is critical in breast physiology; low expression is associated with various aspects of breast cancer. We have found that ionizing radiation induces the rapid expression of M6P/IGF2r in a dose-dependent manner in MCF7 human breast cancer cells. We show that this increase is mediated, at least in part, by a stabilization of M6P/IGF2r transcripts by radiation in both ER positive (MCF7 and T47D) and ER negative (MDA-MB-231) breast cancer cell lines. It is probable, therefore, that posttranscriptional dysregulation of M6P/IGF2r is a contributing mechanism in breast cancer development and breast cancer response to therapy. This is a novel find that underscores the importance of posttranscriptional control of radiation-induced gene expression-a phenomenon that has often been paradigmatically attributed to transcriptional control.

Up-Regulation of Cation-Independent Mannose 6-Phosphate Receptor and Endosomal-Lysosomal Markers in Surviving Neurons after 192-IgG-Saporin Administrations into the Adult Rat Brain

The cation-independent mannose 6-phosphate receptor (CI-MPR) is a single transmembrane domain glycoprotein that plays a major role in the trafficking of lysosomal enzymes from the trans-Golgi network to the endosomal-lysosomal (EL) system. Because dysfunction of EL system is associated with a variety of neurodegenerative disorders, it is possible that the CI-MPR may have a role in regulating neuronal viability after toxicity/injury. In the present study, we report that 192-IgG-saporin-induced loss of basal forebrain cholinergic neurons causes a transient up-regulation of CI-MPR protein levels in surviving neurons of the basal forebrain and frontal cortex but not in the brainstem region, which was relatively spared by the immunotoxin. This was accompanied by a parallel time-dependent increase in other EL markers, ie, cathepsin D, Rab5, and LAMP2 in the basal forebrain region, whereas in the frontal cortex the levels of cathepsin D, and to some extent Rab5, were increased. Given the critical role of the EL system in the clearance of abnormal proteins in response to changing conditions, it is likely that the observed increase in the CI-MPR and components of the EL system in surviving neurons after 192-IgG-saporin treatment represents an adaptive mechanism to restore the metabolic/structural abnormalities induced by the loss of cholin-ergic neurons.

Loss of heterozygosity of M6P/IGF2R gene is an early event in the development of prostate cancer

The genetic events leading to initiation and/or progression of prostate cancer are not well characterized. The gene coding for the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) has recently been identified as a tumor suppressor in several types of cancer. The purpose of the present study is to determine whether the M6P/IGF2R gene is inactivated in human prostate cancer, and if so, whether this is an early or late transformational event. In total, 43 patients with prostate cancer treated by radical prostatectomy, with archival material available for analysis, were assessed for loss of heterozygosity (LOH) in the M6P/IGF2R gene using six different gene-specific nucleotide polymorphisms. Regions of tumor, normal prostate and premalignant high-grade prostate intraepithelial neoplasia (PIN) were identified and cells were excised by laser capture microdissection (LCM). DNA segments were amplified using polymerase chain reaction (PCR). The M6P/IGF2R gene was polymorphic in 83.7% (36/43) of patients, and 41.7% (15/36) of these informative patients had LOH in the tumor tissue. In 11/15 patients with LOH in malignant tissue, high-grade PIN could be identified, and 63.6% (7/11) also had LOH in this premalignant tissue. This study is the first to find that the M6P/IGF2R gene is inactivated in prostate cancer. LOH in premalignant tissue as well suggests that mutation in the M6P/IGF2R gene is an early event in the development of prostate cancer, supporting the conclusion that it functions as a tumor suppressor gene in this disease.

Tissue-Specific Inactivation of Murine M6P/IGF2R

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and T cell- mediated immunity. M6P/IGF2R is an imprinted gene in mice with expression only from the maternal allele. Complete knockout of this gene causes neonatal lethality, thus preventing analysis of its multifunctional role postnatally. To help elucidate the biological functions of M6P/IGF2R in adulthood, we generated both complete and tissue-specific M6P/IGF2R knockout mice using the Cre/loxP system. We confirm that complete M6P/IGF2R knockout results in fetal overgrowth and neonatal lethality. In contrast, tissue-specific inactivation of this gene in either the liver or skeletal and cardiac muscle gives rise to viable animals with no obvious phenotype. The successful creation of viable tissue-specific M6P/IGF2R knockout mouse models will now allow for detailed analysis of receptor function in a number of cellular processes including brain development, carcinogenesis, lysosomal trafficking, and T cell-mediated immunity.

Monotreme IGF2 expression and ancestral origin of genomic imprinting

IGF2 (insulin-like growth factor 2) and M6P/IGF2R (mannose 6-phosphate/insulin-like growth factor 2 receptor) are imprinted in marsupials and eutherians but not in birds. These results along with the absence of M6P/IGF2R imprinting in the egg-laying monotremes indicate that the parental imprinting of fetal growth-regulatory genes may be unique to viviparous mammals. In this investigation, we have cloned IGF2 from two monotreme mammals, the platypus and echidna, to further investigate the origin of imprinting. We report herein that like M6P/IGF2R, IGF2 is not imprinted in monotremes. Thus, although IGF2 encodes for a highly conserved growth factor in chordates, it is only imprinted in therian mammals. These findings support a concurrent origin of IGF2 and M6P/IGF2R imprinting in the late Jurassic/early Cretaceous period. The absence of imprinting in monotremes, despite apparent interparental conflicts over maternal-offspring exchange, argues that a fortuitous congruency of genetic and epigenetic events may have limited the phylogenetic breadth of genomic imprinting to therian mammals. J. Exp. Zool. (Mol. Dev. Evol.) 291:205-212, 2001.

Imprint status of M6P/IGF2R and IGF2 in chickens.

Genomic imprinting is a method of gene regulation whereby a gene is expressed in a parent-of-origin-dependent fashion; however, it is hypothesized that imprinting should not occur in oviparous taxa such as birds. Therefore, we examined the allelic expression of two genes in the chicken that are reciprocally imprinted in most mammals, mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) and insulin-like growth factor 2 (IGF2). Single nucleotide polymorphisms were identified in these genes, and cDNA was prepared from several tissues of embryos heterozygous for these polymorphisms. Both alleles of M6P/IGF2R and IGF2 were expressed in all tissues examined by RT-PCR. Since the expression of these genes was independent of the parent from which they were inherited, we conclude that neither M6P/IGF2R nor IGF2 are imprinted in the chicken.

Mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) variants in American and Japanese populations.

M6P/IGF2R encodes a multifunctional protein involved in lysosomal enzyme trafficking, fetal organogenesis, tumor suppression, and cytotoxic T cell-induced apoptosis. M6P/IGF2R is imprinted and expressed only from the maternally inherited allele in marsupials and rodents. In contrast, humans were initially reported to differ from the imprinted mammalian orders by not having an imprinted M6P/IGF2R; however, some studies now suggest M6P/IGF2R imprinting may be a human polymorphic trait. Mutational and functional evidence are consistent with M6P/IGF2R also being a tumor suppressor in human colon, liver, lung, breast, and ovarian cancers. M6P/IGF2R expression is also pathologically downregulated following mammalian in vitro embryo culture, resulting in fetal overgrowth and "large offspring syndrome." Therefore, the M6P/IGF2R imprint status in humans is an unresolved question that critically impacts upon biological issues ranging from human cancer predisposition to evolution. Attempts to further characterize the imprint status of human M6P/IGF2R and loss of heterozygosity at this locus in cancer have been hindered by a lack of readily usable polymorphisms. To facilitate these genetic analyses, we have screened American and Japanese populations for M6P/IGF2R single nucleotide polymorphisms (SNPs). We have identified nine novel SNPs intragenic to human M6P/IGF2R, and have described experimental conditions for their optimal use. Three identified amino-acid variants in the M6P/IGF2R ligand-binding domains may be under selection in humans.

Loss of heterozygosity at the mannose 6-phosphate insulin-like growth factor 2 receptor ( M6P/IGF2R) locus predisposes patients to radiation-induced lung injury

Purpose: To investigate the relationship between loss of heterozygosity (LOH) at the mannose 6-phosphate/insulin-like growth factor 2 receptor ( M6P/IGF2R) gene locus and the development of radiation-induced lung injury. Material and Methods: Thirty-five lung cancer patients with both stored plasma for Transforming Growth Factor β1 (TGFβ1) analysis and sufficient quantities of archival pathology tissue to screen for LOH were studied. All patients had been treated with thoracic radiotherapy for their malignancy and had radiographically detectable tumor present before beginning radiotherapy. Tumor and normal cells were microdissected from archival lung cancer pathology specimens. Two polymorphisms in the 3′ untranslated region of the M6P/IGF2R were used to screen for LOH. Plasma TGFβ1 levels were measured using acid-ethanol extraction and an ELISA. TGFβ1 and M6P/IGF2R protein expression was estimated by immunofluorescence and immunohistochemical staining. Symptomatic radiation pneumonitis was scored according to National Cancer Institute Common Toxicity Criteria without knowledge of the results of TGFβ or LOH analyses. Results: Of the 35 patients, 10 were homozygous for this polymorphism (noninformative) and were excluded. Of the 25 informative patients, 13 had LOH. Twelve of 13 patients with LOH had increased pretreatment plasma TGFβ1 levels, vs. 3/12 patients without LOH ( p < 0.01). A decrease or loss of M6P/IGF2R protein in the malignant cell accompanied by increased latent TGFβ1 protein in extracellular matrix and tumor stroma was found in tumors with LOH, suggesting that this mutation resulted in loss of function of the receptor. Seven of 13 (54%) LOH patients developed symptomatic radiation-induced lung injury vs. 1/12 (8%) of patients without LOH ( p = 0.05). Conclusions: Loss of the M6P/IGF2R gene strongly correlates with the development of radiation pneumonitis after thoracic radiotherapy (RT). Furthermore, patients with LOH (in the setting of measurable tumor) are much more likely to have elevated plasma TGFβ, suggesting an inability to normally process this cytokine. Thus, loss of the M6P/IGF2R gene may predispose patients to the development of radiation-induced lung injury.

Mutation analysis of the gene encoding the human mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) in human cell lines resistant to growth inhibition by transforming growth factor β 1 (TGF-β 1)

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is involved in activating the transforming growth factor β 1 (TGF-β 1), an inhibitor of the cell proliferation, and limiting the insulin-like growth factor 2 mediated-growth stimulation. The M6P/IGF2R gene has been reported to be mutated and deleted in various cancers, and is a candidate tumor suppressor gene. We studied the genomic structure of the M6P/IGF2R gene and designed the intron primers to detect mutations in the M6P/IGF2R gene of genomic DNA samples. The M6P/IGF2R gene consists of 48 exons. The previously reported 23 mutations of the M6P/IGF2R gene in human cancers, liver, breast, and gastrointestinal tumors, are located in five exons, exon 27, 28, 31, 40, 48. Using the intron primers designed in this study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, and direct sequencing, we performed an initial analysis of the complete coding sequences of the M6P/IGF2R gene in 21 human cell lines resistant to growth inhibition by TGF-β 1. An adenine-to-guanine transition, resulting in an asparagine-to-serine amino acid substitution, was found in one lung adenocarcinoma cell line at exon 40 where the mutation has been previously reported in human cancers. This is the first report of a mutation of the M6P/IGF2R gene in lung tumor. These results indicated that the mutation in M6P/IGF2R may be involved in human lung cancinogenesis.

Mannose 6-phosphate/insulin-like growth factor 2 receptor, a bona fide tumor suppressor gene or just a promising candidate?

The mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) is considered a "candidate" tumor suppressor gene. This hypothesis has been provoked by the identification of loss of heterozygosity (LOH) at the M6P/IGF2R locus on chromosome 6q26 in breast and liver cancer, accompanied by point mutations in the remaining allele. Somatic mutations in coding region microsatellites have also been described in replication error positive (RER+) tumors of the gastrointestinal tract, endometrium and brain. These genetic data are compelling, but a tumor suppressor gene candidate has to meet functional as well as genetic criteria. This review weighs the evidence and discusses the observations that are necessary to promote M6P/IGF2R from candidate to bona fide tumor suppressor gene.

Investigation of possible involvement of several genes related to development of hepatocarcinogenesis in rats.

A comparative study on the possible involvement of several genes in the susceptibility of chemical carcinogenesis was carried out using carcinogen-resistant DRH rat and -sensitive Donryu and F344 rats. Previously, we observed that the induction of glutathione S-transferase placental form (GST-P) in the liver of Donryu rats by 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) was significantly greater than that of DRH rats. In the present study, we tentatively determined base sequences of the enhancer region including GPE-I and GPE-II (GST-P enhancers I and II) of GST-P genes of DRH, Donryu and F344 rats, but we did not observe any nucleotide polymorphism around these regions. Furthermore, the mRNA levels of silencer binding protein (NFA-1) for the GST-P promoter of rat liver were also similar in the DRH and Donryu rats. Since clonal expansion of putative preneoplastic GST-P-positive foci in the DRH rat liver was significantly suppressed during 3'-Me-DAB administration, we examined whether two opposite growth controlling factors, TGF-alpha and TGF-beta, may participate in such suppression of growth. It was supposed that mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R), at least in part, activates TGF-beta preproprotein. However, we observed that the levels of M6P/IGF2R mRNA in the livers of DRH were not higher than those of Donryu rats after being fed 3'-Me-DAB for 8 weeks. Another important factor in the carcinogenesis is insulin-like growth factor II itself. Although liver tumors induced by 3'-Me-DAB in F344 had high levels of IGF-II mRNA, little IGF-II gene expression existed in normal adult livers of Donryu, F344 and DRH rats. High levels of IGF-II mRNA were detected similarly in the livers of neonates from all these three strains of rats. Finally, we detected a significant increase of AFP (alpha-fetoprotein) mRNA in the livers of Donryu rats around 6 to 8 weeks from the start of 3'-Me-DAB feeding, which is in parallel with detrimental effects of this carcinogen on these rats. A reduced induction of AFP mRNA was observed in DRH rats under the same conditions. Further study will be needed to explain the lower tumor susceptibility in the DRH rat.

Failure to detect genetic alteration of the mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) gene in hepatocellular carcinomas in Japan.

The mannose-6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R) suppresses cell growth through binding to the insulin-like growth factor 2 (IGF2) and latent complex of the transforming growth factor-beta (TGF-beta). Recently, it was reported in the United States that loss of heterozygosity (LOH) and mutations in exons 27, 28, and 31 of the M6P/IGF2R gene are frequent in hepatocellular carcinomas (HCCs) and adenomas. In view of the possible importance of this finding, especially for differential diagnosis of small hepatic lesions, we analyzed 43 primary HCCs, 2 adenomatous hyperplasias (AHs), and 3 regenerative nodules (RNs) developing in 42 Japanese patients in Japan for LOH using the polymorphic locus and for mutations by both single strand conformation polymorphism (SSCP) and direct sequencing methods. In the LOH study, 21 out of 22 informative HCCs and all of the informative AHs and RNs showed no allelic loss. In mutational studies of exons 27, 28, and 31, no mutations were detected either by SSCP or direct sequencing analysis in any of the 48 lesions. Thus inactivation of the M6P/IGF2R gene because of genetic alteration does not appear to be essential for hepatocarcinogenesis in Japan.