Midkine Gene Expression Research Articles

Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest

Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised individuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, − 8, and − 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment.

Effect of retinoic acid on midkine gene expression in rat anterior pituitary cells.

Retinoic acid (RA) is converted from retinal by retinaldehyde dehydrogenases (RALDHs) and is an essential signaling molecule in embryonic and adult tissue. We previously reported that RALDH1 was produced in the rat anterior pituitary gland and hypothesized that RA was generated in the gland. Midkine (MK) is an RA-inducible growth factor, and MK production in the rat anterior pituitary gland was recently reported. However, the mechanism that regulates gene expression of MK in the pituitary gland has not been determined. To investigate regulation of MK production in the anterior pituitary gland, we analyzed changes in MK mRNA in cultured rat anterior pituitary cells. We identified MK-expressing cells by double-staining with in situ hybridization and immunohistochemical techniques for RALDH1. MK mRNA was expressed in RALDH1-producing cells in the anterior pituitary gland. Using isolated anterior pituitary cells of rats, we examined the effect of RA on gene expression of MK. Quantitative real-time PCR revealed that 72 h exposure to a concentration of 10-6 M of retinal and all-trans retinoic acid increased MK mRNA levels by about 2-fold. Moreover, the stimulatory effect of all-trans retinoic acid was mimicked by the RA receptor agonist Am80. This is the first report to show that RA is important in regulating MK expression in rat anterior pituitary gland.

Midkine in host defence.

Midkine (MK) shares several features in common with antibacterial proteins of the innate immune system. These include growth factor properties, heparin-binding regions and effects on immune cells, such as recruitment and activation of neutrophils and macrophages. Indeed, recent research has demonstrated potent bactericidal and fungicidal activities of MK. This protein is constitutively expressed at relevant concentrations at barriers of the body, such as the skin and the large airways, where the body first encounters potential pathogens. The antibacterial properties of MK orthologues are preserved during evolution, as exemplified by miple2 of Drosophila. In addition to retinoic acid, promoters of MK gene expression include factors present at sites of infection, reactive oxygen species, activation of the transcription factor NF-κB and hypoxia. In the light of the development of resistance in pathogenic bacteria to conventional antibiotics, MK is an interesting molecule that could serve as a template in developing novel therapeutic strategies against bacterial and fungal infections, either alone or in combination with conventional antibiotics. This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.

The expression of Midkine gene in patients with acute myeloid leukemia and its significance

Midkine (MK) is a heparin-binding growth factor and a product of a retinoic acid-responsive gene. Midkine is overexpressed in many carcinomas and thought to play an important role in carcinogenesis. However, no studies have been focused on the role of MK in AML. This study sought to evaluate the expression of MK among AML patients to explore possible correlation between MK expression and different clinicopathologic variables in an attempt to determine the prognostic value of MK expression. Mononuclear cells were separated from samples of 50 newly diagnosed cases of AML; 10 healthy volunteers were included in this study as control group. The reverse transcription polymerase chain reaction method was used to examine the expression of MK mRNA in mononuclear cells. Midkine gene mRNA expression rate was significantly higher in AML patients when compared to control group (p 0.05). Moreover, Midkine expression was significantly higher in patients that did not achieve complete remission compared to those who succeeded to achieve one (p < 0.001). Midkine may play an important role in the tumorigenesis of AML, and evaluation of MK expression may prove to be useful for predicting response to treatment.

The site-directed mutagenesis and prokaryotic expression of midkine gene in Cynoglossus semilaevis

Midkine is a kind of heparin-binding cytokine,and it promotes growth,survival,migration and other activities of target cells.The expression of fusion protein MK will facilitate further studies for its biological functions in half-smooth tongue sole(Cynoglossus semilaevis).Based on the plasmid containing full length of half-smooth tongue sole MK cDNA sequence,the site-directed mutagenesis of predicted mature MK sequence was successfully obtained by SOEing PCR method according to the E.coli codon bias.After being confirmed by sequencing,the resulting gene was cloned to the vector pET32a(+)to yield an identified recombinant plasmid pET32a(+)-MK,which was then transformed into E.coli host strain BL21(DE3)pLysS.SDS-PAGE analysis revealed that high level of fusion protein MK was induced by 1 mmol/L IPTG at 37 ℃ and was mainly detected in sonicated supernatant.The yielded fusion protein MK was approximately 33 ku which was in accordance with the theoretical molecular weight.This study establishes the basis for further study for its detailed functions in half-smooth tongue sole.

Down-regulation of midkine gene expression by small interfering RNA affects melanoma cell adhesion and invasion

Objective To study the effects of midkine (MK) gene-targeting small interfering RNA (siRNA)on the invasion of melanoma cells.Methods Three MK gene-targeting siRNAs (S1,S2 and S3)were designed,constructed,and transfected into human A375 melanoma cells.Real-time PCR was performed to measure the expression of MK gene and to screen the siRNA with best efficacy.Then,A375 cells were transfected with the optimal siRNA of various doses (3.125,6.25 and 12.5 nmol/L)followed by additional culture of various durations(24,48,72 hours).Some A375 cells remaining untreated served as the blank control group,and some transfected only with liposomes served as the vector control group.Reverse transcription (RT) -PCR and Western blot were conducted to detect the mRNA and protein expression of MK,respectively,MTT assay to observe the adhesion of A375 cells,and Boyden chamber was used to evaluate cell invasion.Results The expression of MK mRNA was downregulated by all the three siRNAs,especially by the siRNA S3,which was used in the following transfection experiment.Real-time quantitative PCR revealed that the MK mRNA expression was reduced by the siRNA in a dose- (r24hours=-0.906,r4Bhours=-0.922,r72hours=-0.939,all P<0.01)and time-dependent(r3.125nmol/L=-0.889,r625nmol/L=-0.935,r125nmol/L=-0.928,all P<0.01)manner.MTT assay showed that the percentage of adhesing cells was 73.66%±2.25%,49.36%±2.16%and 28.35%±1.68%in A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively.The number of cells migrating across the chamber filter was 23.9±1.6,12.1±1.5,5.6±1.2 among A375 cells transfected with the siRNA of 3.125,6.25 and 12.5 nmol/L,respectively,significantly lower than that in the blank control group(36.8±1.5).The percentage of adhesing cells and number of migrating cells decreased with the dose of siRNA(r=-0.936,-0.915,P<0.01,0.05,respectively).Conclusions MK gene might play an important role in the adhesion and invasion of melanonla cells.To down-regulate the expression of MK gene by siRNA may suppress the adhesion and invasion of melanoma cells. Key words: Melanoma,experimental; Cell line,tumor; Midkine; RNA,small interfering

Downregulation of Midkine gene expression and its response to retinoic acid treatment in the nitrofen-induced hypoplastic lung

Nitrofen-induced congenital diaphragmatic hernia (CDH) model has been widely used to investigate the pathogenesis of pulmonary hypoplasia (PH) in CDH. Recent studies have suggested that retinoids may be involved in the molecular mechanisms of PH in CDH. Prenatal treatment with retinoic acid (RA) has been reported to improve the growth of hypoplastic lung in the nitrofen CDH model. Midkine (MK), a RA-responsive growth factor, plays key roles in various organogenesis including lung development. In fetal lung, MK mRNA expression has its peak at E13.5-E16.5 and is markedly decreased during mid-to-late gestation, indicating its important role in early lung morphogenesis. We designed this study to investigate the hypothesis that the pulmonary MK gene expression is downregulated in the early lung morphogenesis in the nitrofen-induced PH, and to evaluate the effect of prenatal RA treatment on pulmonary MK gene expression in the nitrofen-induced CDH model. Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Fetal lungs were harvested on D15, D18, and D21 and divided into control, nitrofen with or without CDH [CDH(+) or CDH(-)]. In addition, RA was given on days D18, D19, and D20 and fetal lungs were harvested on D21, and then divided into control + RA and nitrofen + RA. The pulmonary gene expression levels of MK were evaluated by real-time RT-PCR and statistically analyzed. Immunohistochemistry was also performed to examine protein expression/distribution of MK in fetal lung. The relative mRNA expression levels of MK were significantly downregulated in nitrofen group compared to controls at D15 ((§)p < 0.01), whereas there were no significant differences at D18 and D21. MK gene expression levels were significantly upregulated in nitrofen + RA (0.71 ± 0.17) compared to the control (0.35 ± 0.16), CDH(-) (0.24 ± 0.15), CDH(+) (0.39 ± 0.19) and control + RA (0.47 ± 0.13) (*p < 0.05). Immunoreactivity of MK was also markedly decreased in nitrofen lungs compared to controls on D15, and increased in nitrofen + RA lungs compared to the other lungs on D21. Downregulation of MK gene on D15 may contribute to primary PH in the nitrofen CDH model by disrupting early lung morphogenesis. Upregulation of MK gene after RA treatment in the nitrofen-induced hypoplastic lung suggests that RA may have a therapeutic potential to rescue PH in CDH through RA-responsive growth factor signaling.

Increased drug efflux along with midkine gene high expression in childhood B-lineage acute lymphoblastic leukemia cells

Multidrug resistance (MDR) induced by drug efflux has been identified as the major clinical obstacle in the treatment of childhood acute lymphoblastic leukemia (ALL). To explore the possible effects of midkine (MK) gene on the chemotherapeutic drugs efflux, we examined the MK gene expression in 139 B-lineage ALL patients and 15 children with nonmalignant hematological diseases. Meanwhile, we detected the degree of drug accumulation in 30 progressing B-lineage ALL patients and the 15 control individuals (as control). The results showed that the significant statistic difference in MK gene expression was found among the normal group, the complete remission (CR), and progressing B-lineage ALL patients (p < 0.01). In the Rhodamine 123 (Rh123) efflux test, mean fluorescence intensity (MFI) in the leukemia cells was obviously lower than that in normal pre-B cells (p < 0.01). Furthermore, there was an evident negative correlation between the MFI and MK mRNA expression (r = -0.869, p < 0.001) but no correlation with the MDR1 mRNA expression (p > 0.05). We concluded that there was powerful drug efflux ability in lymphoblastic leukemia cells with high MK gene expression. MK gene may take part in multidrug resistance.

Expression of Midkine(MDK) and Sensitivity to Anti-Leukemic Drugs in Pediatric Acute Lymphoblastic Leukemia (ALL).

Midkine (MDK), a heparin-binding growth factor, is highly expressed in pediatric ALL, neuroblastomas, and various solid tumors. Previous gene expression profiling studies conducted in our laboratory on a cohort of 220 children with B precursor ALL treated on Children's Oncology Group (COG) clinical trials have shown that higher levels of MDK expression in leukemic blasts are strongly associated with continuous complete remission (CCR) and that MDK is a powerful prognostic predictor of outcome (Wilcoxon ranksum test, p=0.000001), particularly in patients with central nervous system (CNS) involvement (Wilcoxon ranksum test, p =0.009). To explore the role of MDK in hematopoiesis and leukemogenesis and to further explore MDK as a potential novel target for ALL therapy, we examined MDK mRNA expression in three stages of normal B cell development in healthy controls and compared it to expression levels in leukemic blasts from children with B-ALL. Quantitative RT-PCR assays showed that MDK mRNA levels in CD19+ cells from peripheral blood mononuclear cells (PBMNC) were significantly higher than those in CD34+ or CD19+ cells from bone marrow (BM) (Mann Whitney test, p=0.0025 and 0.0018, respectively). ALL leukemic blasts samples expressed significantly higher levels of MDK than those in CD19+ cell in the BM of healthy controls (Mann Whitney test, p=0.0043). Similar results were obtained by ELISA detection of MDK protein in plasma of ALL patients and healthy controls. We have also investigated the relationship of MDK expression and the chemo-sensitivity of ALL cell lines. Using a lentiviral system, we have successfully over-expressed or knocked down MDK gene expression in the REH cell line containing the t(12;21). Unexpectedly, MTS assays revealed that MDK expression is inversely associated with sensitivity to L-asparaginase and doxorubicin, but not to vincristine and cytarabine, in REH cells. Higher levels of MDK were associated with chemo-resistance. Finally, we tested the effects of MDK expression on proliferation and apoptosis in REH cells and determined that although MDK expression was not associated with REH cell proliferation in vitro, it protected REH cells from doxorubicin-induced apoptotic cell death. Taken together, these results suggest that expression levels of MDK in ALL is inversely associated with in vitro drug sensitivity to specific anti-leukemic agents; consistent with observations in solid tumor models. It has been suggested that 4β1- and 6β1-integrins bind directly to MDK and are involved in MK-dependent cell migration. Further studies of the role of MDK signaling in vivo in the complex marrow/adhesion microenvironment and the effects on ALL survival and chemo-sensitivity and resistance are in progress.

Quantification of midkine gene expression in Patella caerulea (Mollusca, Gastropoda) exposed to cadmium

The release of cadmium into many coastal areas represents a threat to ecosystems and human health; cadmium is carcinogenic in mammals and in both marine invertebrates and vertebrates. The use of molluscs to assess the ecologic risk associated with contaminants is strongly recommended on account of their ecological role and on their highly conserved control and regulatory pathways that are often homologous to vertebrate systems. We previously identified a midkine family protein in the limpet Patella caerulea; the midkine is a recently discovered cytokines family with unequivocal informative value on repairing injury and neoplastic processes in mammals. Here we report on midkine ( mdk) and α-tubulin ( α-tub) gene expression patterns in P. caerulea exposed to cadmium. Limpets, collected on two occasions from a breakwater at a marina (Tyrrhenian Sea) were exposed to sublethal cadmium concentrations (0.5 and 1 mg l −1 Cd) over a 10-day exposure period. RNA was extracted from the viscera of unexposed and exposed specimens. Real time TaqMan RT-PCR was performed to measure the relative mdk and α-tub gene expression levels. A remarkable mdk over-expression was observed in all exposed animals with respect to unexposed ones; mdk over-expression was significantly higher in both treatments when compared with un-treatment (mean expression levels: 23- and 38-fold, for 0.5 and 1 mg l −1 Cd treatment, respectively; ANOVA, for both P < 0.01). The study also indicates that the mdk up-regulation was significantly Cd-concentration dependent ( P < 0.05). A significant up-regulation of the constitutive α-tub gene was also observed in 1 mg l −1 Cd-treated animals (mean expression level: 4-fold; ANOVA, P < 0.05). In conclusion, these data provide the first evidence paving the way for the use of the midkine as a promising new biomarker of effect in the environment risk assessment policy.

Increased midkine gene expression in childhood B-precursor acute lymphoblastic leukemia

Midkine (MK) is a heparin-binding growth factor that is overexpressed in a number of solid cancers. However, expression in acute leukemia has not been clarified. We examined MK gene expression using real-time PCR in 94 children with acute leukemia. In 30 of the 41 patients with B-precursor ALL, MK gene expression was overexpressed than normal BM. MK gene was also overexpressed in more than half of patients with FAB M1 and M2 types of AML. Quantification of MK gene by real-time PCR offers particular promise as a prognostic marker and a marker for minimal residual disease in children with B-precursor ALL.

Increased Midkine Gene Expression in Childhood Acute Leukemia.

Midkine (MK) is a heparin-binding growth factor and overexpressed in a number of solid tumors, contributing to their growth. Its expression in acute leukemia, however, has not been clarified. We examined relative levels of MK gene expression defined in K562 cells as 1.00 using real-time quantitative polymerase chain reaction (PCR) in 94 children with acute leukemia and compared with those in normal bone marrow (BM; n=18) and peripheral blood (PB; n=10). Median age was 6 years (range, 0–15 years). Diagnosis included B-precursor ALL (n=41), T-ALL (n=14), B-ALL (n=4), Ph1-ALL (n=6), infant ALL (n=5) and AML (n=24). 8 normal BM samples were separated into CD34+ and CD34− fractions. MK gene expression was detected in normal control samples. Median MK gene expression level was significantly lower in normal PB (2.9×10e-3) than in normal BM (8.0×10e-3) (p<0.01). Expression of MK gene was higher in normal CD34+ BM cells (0.78×10e-3) than in normal unfractionated BM cells (p<0.001). Overexpression of MK was difined as expression at least twice as high as the maximal MK gene expression in normal PB and BM. In 30 of the 41 patients with B-precursor ALL (73.2%), MK gene is overexpressed. We classified B-cell lineage ALL into 3 stage:DR+,CD19+,CD10+,CD20−,slg−,DR+,CD19+,CD10+,CD20+,slg−,DR+,CD19+,CD20+,slg+.MK gene expression decreased with B-cell lineage differentiation of leulkemic blast. It was also overexpressed in more than half of the patients with FAB M1 and M2 types of AML. In contrast, overexpression of MK gene was observed only in one of the 13 patients with T-ALL and none of the 6 patients with AML-M5. Supporting the possible involvement of MK in carcinogenesis, overexpression of MK may be not only merely assoziated with total development but also related to leukemic transformation of hematopoietic progenitors. Quantification of MK gene by real-time PCR is relatively simple and widely applicable in patients with acute leukemia. This technique offers particular promise as a prognostic marker and a marker for minimal residual disease in children with B-precursor ALL.

Morphine and yohimbine regulate midkine gene expression in the rat hippocampus

Pleiotrophin and midkine are two recently discovered growth factors that promote survival and differentiation of catecholaminergic neurons. Chronic opioid stimulation has been reported to induce marked alterations of the locus coeruleus–hippocampus noradrenergic pathway, an effect that is prevented when opioids are coadministered with the α 2-adrenoceptor antagonist yohimbine. The present work tries to examine a possible link between yohimbine reversal of morphine effects and pleiotrophin/midkine activation in the rat hippocampus by studying the levels of expression of pleiotrophin and midkine in response to acute and chronic administration of morphine, yohimbine and combinations of both drugs. Pleiotrophin gene expression was not altered by any treatment; however midkine mRNA levels were increased after chronic treatment with morphine. Chronic administration of yohimbine alone also increased midkine expression levels, whereas yohimbine and morphine administered together exhibited summatory effects on the upregulation of midkine expression levels. The data suggest that midkine could play a role in the prevention of opioid-induced neuroadaptations in hippocampus by yohimbine.

Midkine Expression in Reed-Sternberg Cells of Hodgkin's Disease

Midkine (MK) was originally cloned as a product of a retinoic acid-responsive gene. The rationale for studying MK expression is based on previous reports showing that it transforms 3T3 cells, and that it acts as an autocrine growth factor in Wilm's tumors, and that its overexpression has been associated with worse outcome in bladder carcinoma. Besides bladder carcinoma, its expression was reported in various sollid tumors. We investigated the expression of MK protein and/or MK gene in biopsied specimens from 40 patients with primary malignant lymphoma, 21 with Hodgkin's disease (HD) and 19 with non-Hodgkin's lymphoma (NHL). Reed-Sternberg (R-S) cells were stained positive in 10 of 16 HD cases evaluated by immunohistochemical method, whereas 18 of 19 NHL cases did not stain, and one B-cell NHL stained weakly positive. Immunostaining analysis was extended to established cell lines and to normal lymphocytes with or without lectin stimulation or with EB virus transformation. Among hematopoietic ceils examined, erythro- or megakaryoblastic leukemia cell lines (K562, MEG-01 and UT7) were positive, while normal lymphocytes (except the EB virus-transformed one) and most myeloid and lymphoid cell lines (except Raji cells) were negative. On the contrary, solid tumor cell lines showed high and strongly positive staining including cell lines derived from of lung gastric, colon, and a pancreatic cancer.Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), which is suitable for the detection of weakly expressed mRNA, the relative ratio of MK mRNA to β-actin mRNA of samples was measured and compared in cases where RNA was available. The mean values of relative ratio (MK/β-actin) of HD were almost twice as those of NHL samples, peripheral blood T cells, and spleen B cells. Our findings showed that MK is expressed in Reed-Sternberg cells of HD, and that MK might play a role in the pathogenesis of HD.

Midkine gene expression in the healing process of gastric ulcer

Midkine is a newly cloned growth-promoting factor for fibroblastic cells. This study was performed to investigate the possible role of midkine in the stomach. Rats with acute or chronic gastric mucosal lesions were used. Histologically, acute mucosal lesions are not accompanied by the formation of granulation tissue; conversely, chronic mucosal lesions are accompanied by the formation of granulation tissue. The expression of the midkine gene was found in the normal intact gastrointestinal tract, especially the submucosal and muscle layers. Midkine mRNA increased during the healing stage of chronic gastric ulcer accompanied by a fibroblastic reaction. Furthermore, the fibroblast cell line MRC-5 expressed midkine mRNA strongly. Therefore, midkine may have some role in the healing of gastric deep ulcers that is accompanied by fibroblast proliferation.

Frequent expression of midkine gene in esophageal cancer suggests a potential usage of its promoter for suicide gene therapy.

We have examined the expression of midkine (MK), a neurotrophic factor with heparin‐binding activity, in human esophageal cancer cells. Seven esophageal cell lines tested expressed the transcript and 8 out of 14 human esophageal tumor specimens were positively stained with anti‐MK antibody, while surrounding normal esophageal tissues in these specimens were not stained. The 5′‐flanking, 2.3 kb genomic region of the MK gene was shown to drive the transcription of a reporter gene in the esophageal cell lines in a cis acting manner. Forced expression in esophageal cancer cells of herpes simplex virus‐thymidine kinase gene mediated by the flanking region of the MK gene conferred sensitivity to a prodrug, ganciclovir. The 5′‐upstream region of the MK gene thus possesses putative promoter activity which can be used for suicide gene‐based gene therapy for esophageal cancer.

Expression of midkine and pleiotropin in ovarian tumors

To compare the expression of midkine and pleiotropin in malignant ovarian tumors with that in normal and benign ovarian tissue. Total RNA was isolated from 23 samples of normal ovaries, 15 benign ovarian tumors, and 36 malignant ovarian tumors. Midkine and pleiotropin gene expression was examined by using Northern blot analysis. To confirm the localization of midkine expression, in situ hybridization and immunohistochemical analyses were performed. The truncated midkine messenger RNA was examined using polymerase chain reaction with complementary DNA synthesized from total RNA with reverse transcriptase. Expression of midkine gene was observed in 19 of 23 normal ovary samples and in 51 of 53 ovarian tumors (13 of 15 benign, both of the two borderline tumors, and all 36 malignant tumors). Pleiotropin gene was expressed in six normal ovaries and in 24 tumors (nine benign, two borderline, and 13 malignant tumors). The expression of midkine in germ cell tumors was significantly lower than in epithelial tumors, whereas expression in malignant epithelial tumors was significantly higher than in benign ones. In germ cell tumors, two samples with differentiated neural tissues showed high levels of pleiotropin gene expression. In situ hybridization and immunohistochemical analysis showed strong expression of midkine in cancer cells. The truncated midkine messenger RNA was not found in any of the normal, benign, or malignant tissues examined. These results suggest an association between midkine and carcinogenesis. Expression of pleiotropin is more restricted, and high levels of its expression may be correlated with neural differentiation.

Increased midkine gene expression in human gastrointestinal cancers.

Midkine (MK) is a product of a retinoic acid‐responsive gene, and is a novel growth differentiation factor. We examined the expression of the MK gene in specimens of 47 surgically removed human carcinomas of the gastrointestinal organs, namely, gastric, colorectal, hepatocellular, pancreatic, esophageal, ampullary duodenal and bile duct carcinomas. In most cases, the MK mRNA level was higher in cancer specimens than in the corresponding non‐cancerous tissues. Furthermore, MK mRNA was more highly expressed in the colon adenocarcinoma lesion than in the adenoma lesions, in the two familial polyposis cases. While MK mRNA was not detected in the normal liver, it became detectable in cirrhotic tissues in 2 of 4 cases, and its expression was increased in the cancerous tissues. Thus, the increase of MK mRNA level is a phenomenon seen in many human gastrointestinal carcinomas. The increased expression of the MK gene in gastric carcinoma was significantly more prominent in well and moderately differentiated adenocarcinomas than in poorly differentiated adenocarcinomas and signet ring cell carcinomas.

Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis.

Midkine (MK) is the first cloned gene in a new family of heparin-binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.

The Upstream Sequence of a New Growth/Differentiation Factor, Midkine (MK), Mediates Developmentally Regulated lac Z Gene Expression in Transgenic Mice.

Midkine (MK) is the product of a retinoic acid responsive gene, and is a heparin binding protein involved in the regulation of growth and differentiation. The 1.9 kb upstream region of MK gene was fused with the bacterial β-galactosidase gene (lac Z) and injected into fertilized mouse eggs. The resulting transgenic mice were used to evaluate the in vivo transcriptional regulation through of the upstream region. Comparison of the β-galactosidase expression and endogenous MK expression indicated that the temporal regulation of the transgene was similar to that of MK gene expression during mouse development. The transgene was neither expressed in the preimplantation period nor in 6.5-day embryos. Transgene expression was high and widely distributed on the 8.5th day, became restricted on the 10.5th and 12.5th days, and thereafter almost confined to the kidney. Thus, the 1.9 kb upstream region accounts for overall temporal regulation of MK gene expression, while there are some differences between the spatial regulation of the transgene expression and that of the endogenous MK gene expression. The transgene was expressed in a few limited regions of the brain of 17 day old embryos, and those sites consisted largely of matrix cells with columnar arrangements. These results suggests a role of MK in the brain development, and MK activity may be involved in retinoic acid induced malformations of the central nervous system.