NF-κB inhibition reveals a novel role for HGF during skeletal muscle repair.

The transcription factor nuclear factor κB (NF-κB)/p65 is the master regulator of inflammation in Duchenne muscular dystrophy (DMD). Disease severity is reduced by NF-κB inhibition in the mdx mouse, a murine DMD model; however, therapeutic targeting of NF-κB remains problematic for patients because of its fundamental role in immunity. In this investigation, we found that the therapeutic effect of NF-κB blockade requires hepatocyte growth factor (HGF) production by myogenic cells. We found that deleting one allele of the NF-κB subunit p65 (p65+/-) improved the survival and enhanced the anti-inflammatory capacity of muscle-derived stem cells (MDSCs) following intramuscular transplantation. Factors secreted from p65+/- MDSCs in cell cultures modulated macrophage cytokine expression in an HGF-receptor-dependent manner. Indeed, we found that following genetic or pharmacologic inhibition of basal NF-κB/p65 activity, HGF gene transcription was induced in MDSCs. We investigated the role of HGF in anti-NF-κB therapy in vivo using mdx;p65+/- mice, and found that accelerated regeneration coincided with HGF upregulation in the skeletal muscle. This anti-NF-κB-mediated dystrophic phenotype was reversed by blocking de novo HGF production by myogenic cells following disease onset. HGF silencing resulted in increased inflammation and extensive necrosis of the diaphragm muscle. Proteolytic processing of matrix-associated HGF is known to activate muscle stem cells at the earliest stages of repair, but our results indicate that the production of a second pool of HGF by myogenic cells, negatively regulated by NF-κB/p65, is crucial for inflammation resolution and the completion of repair in dystrophic skeletal muscle. Our findings warrant further investigation into the potential of HGF mimetics for the treatment of DMD.

Hepatocyte growth factor/scatter factor is a motogen for interneurons migrating from the ventral to dorsal telencephalon.

Cortical interneurons arise from the proliferative zone of the ventral telencephalon, the ganglionic eminence, and migrate into the developing neocortex. The spatial patterns of migratory interneurons reflect the complementary expression of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, MET, in the forebrain. Scatter assays on forebrain explants demonstrate regionally specific motogenic activity due to HGF/SF. In addition, exogenous ligand disrupts normal cell migration. Mice lacking the urokinase-type plasminogen activator receptor (u-PAR), a key component of HGF/SF activation, exhibit deficient scatter activity in the forebrain, abnormal interneuron migration from the ganglionic eminence, and reduced interneurons in the frontal and parietal cortex. The data suggest that HGF/SF motogenic activity, which is essential for normal development of other organ systems, is a conserved mechanism that regulates trans-telencephalic migration of interneurons.

Cationic colloidal silica membrane perturbation as a means of examining changes at the sinusoidal surface during liver regeneration.

By employing the cationic colloidal silica membrane density perturbation technique, we examined growth factor receptor and extracellular matrix (ECM) changes at the sinusoidal surface during rat liver regeneration 72 hours after 70% partial hepatectomy (PHx). At this time after PHx, hepatocyte division has mostly subsided, while sinusoidal endothelial cell (SEC) proliferation is initiating, resulting in avascular hepatocyte islands. Because of the discontinuous nature of the surface of liver SEC, ECM proteins underlying the SEC, as well as SEC luminal membrane proteins, are available to absorption to the charged silica beads when the liver is perfused with the colloid. Subsequent liver homogenization and density centrifugation yield two separate fractions, enriched in SECs as well as hepatocyte basolateral membrane-specific proteins up to 50-fold over whole liver lysates. This technique facilitates examination of changes in protein composition that influence or occur as a result of SEC mitogenesis and migration during regeneration of the liver. When ECM and receptor proteins from SEC-enriched fractions were examined by Western immunoblotting, urokinase plasminogen activator receptor, fibronectin, and plasmin increased at the SEC surface 72 hours after PHx. Epidermal growth factor receptor, plasminogen, SPARC (secreted protein, acidic and rich in cysteine, also called osteonectin or BM40), and collagen IV decreased, and fibrinogen subunits and c-Met expression remained constant 72 hours after PHx when compared to control liver. These results display the usefulness of the cationic colloidal silica membrane isolation protocol. They also show considerable modulation of surface components that may regulate angiogenic processes at the end stage of liver regeneration during the reformation of sinusoids.

Growth factor signal transduction immediately after two-thirds partial hepatectomy in the rat.

Liver regeneration after partial hepatectomy (PHx) of the liver serves as a model for studying normal growth factor signals that become aberrant in cancer. Growth factor signals that may play a role in initiating the proliferation of hepatocytes after 70% PHx in the rat were investigated immediately after surgical resection of the liver. Presumptive activity was evaluated by determining the tyrosine phosphorylation state of receptors for epidermal growth factor (EGF) and hepatocyte growth factor (HGF) in the liver after PHx and after sham operation as a control. Under these conditions, it was determined that the EGF receptor was constitutively phosphorylated. EGF receptor tyrosine phosphorylation, however, was increased over basal levels by 60 min after resection. The HGF receptor, c-Met, was minimally phosphorylated in control livers, but a biphasic increase in phosphorylation was observed at 1-5 min after PHx and 60 min postsurgery. A slight increase in c-Met phosphorylation was observed in the sham-operated livers, but the signal was significantly less when compared with that in resected livers. Furthermore, 1 min after PHx, but not sham operation, urokinase-type plasminogen activator (u-PA) and u-PA receptor were observed in the immunoprecipitates of c-Met. Signaling downstream of growth factor receptor activation was also examined. There were no discernible phosphorylation changes in focal adhesion kinase during the early events after surgery in PHx; however, a rapid and sustained increase in the tyrosine phosphorylation of paxillin beginning 1 min after PHx, and a gradual increase in the phosphorylation beginning 5 min postsham operation, were observed. Changes in the activated state of the small GTP-binding protein Rho A and its associated proteins were seen but only after 3 h after PHx. The results indicate that HGF-related signal transduction cascades, which contribute to hepatocyte proliferation, are initiated within one min after PHx.

Bone marrow as a potential source of hepatic oval cells.

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

Differential expression and distribution of focal adhesion and cell adhesion molecules in rat hepatocyte differentiation.

Hepatocytes in primary culture enter into clonal proliferation in the chemically defined hepatocyte growth medium in the presence of hepatocyte growth factor and epidermal growth factor. Hepatocyte proliferation is associated with loss of differentiated gene expression. Overlay of matrix derived from Engelbreth-Holm-Swarm mouse sarcoma (Matrigel) on proliferating hepatocytes induces reexpression of the hepatic differentiation marker genes. To explore the role of matrix in the differentiation process of hepatocytes, we examined the mRNAs of fibronectin, vitronectin, and entactin in proliferating hepatocytes and Matrigel-treated hepatocytes. Fibronectin mRNA increased in proliferating hepatocytes at days 2-10 and then decreased; however, vitronectin mRNA disappeared in proliferating hepatocytes and was reexpressed in Matrigel-treated hepatocytes. We also found that focal adhesion kinase and paxillin were strongly increased in Matrigel-treated hepatocytes, and E-cadherin and beta-catenin slightly increased in Matrigel-treated hepatocytes, suggesting that both cell-to-extracellular matrix and cell-to-cell interactions may be an essential part of hepatocyte differentiation. To evaluate the distribution of focal adhesion associated molecules and cell-to-cell adhesion molecules, Triton X-100 soluble and insoluble fractions were examined at days 8, 9, 10, and 11 in proliferating hepatocytes and Matrigel-treated cells. We found that E-cadherin in Triton X-100 insoluble fractions dramatically decreased in Matrigel-treated hepatocytes; however, beta-catenin strongly increased in Triton X-100 soluble fractions of Matrigel-treated hepatocytes. These results suggest that the distribution of both focal adhesion associated molecules and cell adhesion molecules are reorganized during the process of differentiation induced by overlay of Matrigel.

Dose-dependent biphasic effects of phenobarbital on growth and differentiation of primary culture rat hepatocytes.

The actions of phenobarbital, a liver tumour promoter, on growth and differentiation of primary culture normal rat hepatocytes change biphasically as a function of its concentration. At low concentrations of 0.5-2 mmol/L, phenobarbital enhances DNA synthesis of normal adult rat hepatocytes in the presence of epidermal growth factor (EGF) and/or dexamethasone. This is also true for normal suckling (1-2-week-old) rat hepatocytes, without added growth factor(s), in serum-free primary culture. Contrarily, phenobarbital at high concentrations (3-4 mmol/L) suppresses DNA synthesis of suckling rat hepatocytes. Furthermore, phenobarbital inhibits DNA synthesis of transforming growth factor-alpha-stimulated primary hepatocytes from normal adult rats in a dose-dependent manner within a concentration range of 3-6 mmol/L. When normal adult rat hepatocytes are led to undergo multiple proliferative cycles upon stimulation with hepatocyte growth factor (HGF) and EGF in the chemically defined hepatocyte growth medium (HGM), 3 mmol/L phenobarbital also remarkably suppresses DNA synthesis. Phenobarbital at 3 mmol/L effectively keeps these hepatocytes morphologically differentiated and accelerates restoration of the expression of markers characteristic of differentiated cells after the initial cellular growth phase. In addition, phenobarbital efficiently supports prolonged survival of the hepatocytes.

Phenobarbital suppresses growth and accelerates restoration of differentiation markers of primary culture rat hepatocytes in the chemically defined hepatocyte growth medium containing hepatocyte growth factor and epidermal growth factor.

Phenobarbital (PB), a liver-tumor promoter, at a concentration of 3 mM dramatically inhibited the growth of adult rat hepatocytes in the chemically defined medium, HGM, with added hepatocyte growth factor (HGF) and epidermal growth factor (EGF). In concurrence with these findings, PB down-regulated expression of the HGF receptor (c-met) and suppressed production of the autocrine growth factor transforming growth factor-alpha (TGF-alpha). Furthermore, PB down-regulated expression of transcription factors associated with proliferation such as AP1 and NF-kappaB. In the presence of PB, hepatocytes remained morphologically differentiated and restoration of the expression of mature hepatocyte markers, such as albumin and cytochrome P450s (1A, 2B1/2, and 2E1), was accelerated after an initial phase of growth. Additionally, PB strongly suppressed expression of the mRNA for alpha-fetoprotein, a protein primarily expressed by fetal liver, and the accelerative effect of PB on restoration of mature hepatocyte markers showed a correlation with the up-regulation of the hepatocyte-enriched transcription factors HNF3 and HNF4. When the effects of PB on various extracellular matrix proteins were examined, the data indicated that PB specifically suppressed laminin and fibronectin production by hepatocytes, suggesting an important role for these proteins in growing hepatocyte cultures.

Modifications of the hepatocyte growth factor/c-met pathway by constitutive expression of transforming growth factor-alpha in rat liver epithelial cells.

We have previously shown that rat liver epithelial cells (RLEC) transfected with and constitutively expressing transforming growth factor-alpha (TGF-alpha) have an enhanced mitogenic response to hepatocyte growth factor (HGF). In the study reported here, we examined tumor clones derived from the TGF-alpha transfectants with respect to mitogenic response to HGF. Tumor cell lines that expressed TGF-alpha responded to HGF with a greater increase in DNA synthesis than did the nontransfected parental RLEC (pRLEC). The tumor clones had also acquired a lower threshold for HGF response, which enabled them to undergo significant DNA synthesis at a low concentration of HGF that did not evoke a response in the pRLEC or TGF-alpha transfectants. We investigated the mechanisms by which TGF-alpha expression may influence the HGF/c-met pathway. We showed that most TGF-alpha transfectants and tumor cells displayed increases in c-met mRNA and protein, indicating that the enhanced HGF response may be due in part to an increase in the amount of receptor present. However, in all transfectants and tumor clones that constitutively expressed TGF-alpha, c-met was tyrosine phosphorylated in the absence of ligand (HGF) or other exogenous growth factors. These data suggest that induction of c-met mRNA and transactivation of c-met may be a sequela of the constitutive expression of TGF-alpha and that constitutive activation of the epidermal growth factor receptor pathway leads to phosphorylation and activation of c-met. These studies provide evidence for a novel mechanism of communication between epidermal growth factor receptor and c-met pathways that may partially explain the synergistic effects reported between TGF-alpha and HGF.

Presence of urokinase in serum-free primary rat hepatocyte cultures and its role in activating hepatocyte growth factor.

Serum-free rat hepatocyte cultures can be stimulated to divide by the inactive, single-chain form of hepatocyte growth factor (scHGF), suggesting that hepatocytes contain a protein that can cleave scHGF to its biologically active, two-chain (tcHGF) form. We added radiolabeled scHGF to serum-free cultures and confirmed that tcHGF was being generated. Because scHGF can be cleaved to tcHGF by plasminogen activators (PAs), we next tested the cultures for active PA. Although little PA activity was initially present, the majority was of the urokinase type (u-PA) as determined by neutralization studies using either a polyclonal antibody against u-PA or, since u-PA functions in the context of its receptor (u-PAR), a monoclonal antibody against u-PAR. Considerable PA activity developed within 24 h, which was also neutralizable with antibody. To test whether the active, receptor-bound u-PA from the cell cultures was cleaving scHGF, iodinated scHGF was added to intact cells in the presence of the antibody against u-PAR. Comparison to control cultures determined that the antibody prevented scHGF cleavage. Analysis of cultures treated with HGF, epidermal growth factor, and transforming growth factor alpha (TGF-alpha) alpha showed these growth factors increased the hepatocyte PA activity in parallel with the mRNA for u-PA. TGF-beta had the opposite effect, and when TGF-beta was added to the culture system, conversion of scHGF to tcHGF was prevented in concert with the production of the type 1 PA inhibitor. When liver remnants from hepatectomized animals were assayed for active TGF-beta, elevated protein was found just prior to the appearance of PA inhibitor 1 message and protein. Collectively, our data show that in culture, active u-PA is present and cleaves scHGF to tcHGF in the context of its receptor. It also suggests that modulation of u-PA activity by various growth factors is relevant for regulating cleavage of scHGF to tcHGF both in vitro and in vivo.

Inheritance of unequal numbers of the genes encoding the human neutrophil defensins HP-1 and HP-3.

It is unclear whether the six known human defensin peptides are all encoded by separate genes or whether some of them are allelic. Three of the peptides, HP-1, HP-2, and HP-3, differ by only one amino acid, and it is thought that HP-2 may represent a proteolytic product of HP-1 and/or HP-3. To help determine the relationship of these three proteins, we isolated a nearly full-length cDNA encoding HP-1 with a sequence very similar to, but different from, the previously isolated HP-1 and -3 cDNAs. Gene copy number experiments established that there were at least two but fewer than five defensin genes with a high level of similarity to the HP-1 cDNA (HP-1/3-like). Three genomic clones were isolated that contained two different configurations of the HP-1/3-like sequences. Sequencing established that one encoded the HP-1 peptide, whereas the other encoded HP-3. Analysis of DNAs obtained from 18 unrelated individuals by Southern blot analysis revealed the expected fragments as well as additional fragments that were not present in the genomic clones. This suggested the possibility of alleles; however, when DNAs from families were examined, these fragments did not segregate in an obvious Mendelian fashion. The HP-1/3-like defensin genes are on human chromosome 8. Surprisingly, somatic cell hybrid mapping showed that the number of HP-1/3-like genes on isolated copies of chromosome 8 was variable. We conclude that individuals can inherit versions of chromosome 8 harboring either two or three copies of the genes that encode the HP-1, HP-2, and/or HP-3 peptides.

Immediate early detection of urokinase receptor after partial hepatectomy and its implications for initiation of liver regeneration.

Hepatocyte growth factor (HGF), also known as scatter factor, is believed to play a primary role in liver regeneration. HGF is produced in an inactive single-chain form that can be cleaved in vitro to the active two-chain form by tissue-type and urokinase-type plasminogen (PLG) activators (tPA and uPA). We have now documented the de novo appearance of active uPA in livers from male Fischer F344 rats that underwent 70% partial hepatectomy (PHx) as early as 1 minute after surgery. Western blot analyses of protein extracts from liver remnants that were obtained immediately after surgery and periodically until 24 hours after PHx indicate that the quantity of uPA remains fairly constant in PHx samples. In contrast, the uPA receptor (uPAR) dramatically increases, beginning within 1 minute after PHx. This results in enhanced activity of uPA, as seen by direct zymography on cryostat sections. The uPA present in remnant liver homogenates from rats that underwent PHx is the primary agent that cleaves single-chain HGF to its two-chain form, because cleavage can be prevented when antibody against uPA is included in the liver homogenates. Furthermore, heterodimeric HGF, which is not present in normal liver, increases in the liver remnants from rats that underwent PHx, correlative to uPAR. The presence of active uPA is one of the earliest responses yet documented after PHx. These findings imply that both uPA and uPAR are involved in activating endogenous HGF in the regenerating livers of animals that underwent PHx.

Hepatocyte growth factor inhibits cell proliferation in vivo of rat hepatocellular carcinomas induced by diethylnitrosamine.

In addition to its mitogenic, motogenic and morphogenic functions on various cell types, hepatocyte growth factor (HGF) also suppresses mitosis in several cancer cell lines, including carcinomas and sarcomas. Here we report that HGF is also mito-inhibitory in rat liver tumors induced by diethylnitrosamine. By using a double labeling technique employing [3H]thymidine and 5-bromo-2'-deoxyuridine to determine cell proliferation before and after HGF infusion, we determined that continuous infusion of 20 micrograms total HGF inhibited tumor cell proliferation by 50%. The labeling in non-tumor areas showed the reverse result in that the labeling was higher in HGF-treated rats than control rats. These results indicate that HGF has different effects on growth of normal and tumorous hepatocytes in vivo. These findings may be of relevance in understanding the role of HGF in hepatocarcinogenesis and provide added modalities for controlling growth of hepatocellular carcinomas.

Activation of hepatocyte growth factor by the plasminogen activators uPA and tPA.

Hepatocyte growth factor, also known as scatter factor, is a complete mitogen for hepatocytes that bears sequence and structural homology with plasminogen. Because it exists in both a mitogenically inactive single-chain form and an active two-chain form, we were interested in determining whether plasminogen activators could properly cleave single-chain hepatocyte growth factor to generate active two-chain hepatocyte growth factor. Herein we report that both urokinase-type plasminogen activator and tissue-type plasminogen activator can cleave single-chain hepatocyte growth factor, generating two-chain hepatocyte growth factor. When equal quantities of plasminogen activator-treated and activator-untreated hepatocyte growth factor are compared in serum-free in vitro bioassays, the treated hepatocyte growth factor is mitotically more active. Also, urokinase-type plasminogen activator was inactive against hepatocyte growth factor molecules with a mutated cleavage site. This suggests that urokinase-type and tissue-type plasminogen activator may be natural biological regulators of hepatocyte growth factor. Because the active form of hepatocyte growth factor is a powerful stimulator of DNA synthesis and cell motility, these findings may be relevant in understanding the role of plasminogen activators in the biology of cancer invasion and metastasis.

Expression of elongation factor-1 gamma-related sequence in human pancreatic cancer.

A cDNA clone designated pPDC-1 was isolated from a cDNA library prepared against poly(A+)RNA isolated from the human pancreatic adenocarcinoma cell line, Capan-2. The cDNA corresponds to a 1.7-kilobase mRNA that is expressed at higher levels in seven of nine pancreatic tumors than in their corresponding normal tissues. It is also expressed in normal human kidney, intestine, pancreas, stomach, placenta, lung, brain, spleen, and liver. A computer search of the Intelligenetics System of all available nucleotide sequences revealed a 60% homology between the nucleotide sequence of the pPDC-1 cDNA and that of elongation factor-1 gamma from Artemia. The deduced amino acid sequence shared 53% identity with the amino acid sequence for the Artemia elongation factor-1 gamma.

Expression pattern of a hematopoietic proteoglycan core protein gene during human hematopoiesis.

Expression of the hematopoietic proteoglycan core protein (HpPG) gene was examined in normal peripheral blood, normal bone marrow, and leukemic peripheral blood leukocytes samples to assess the expression pattern of the HpPG gene in these cells and to ascertain points of regulation of this gene during hematopoiesis. In situ hybridization to normal bone marrow and peripheral blood leukocytes demonstrated that the gene was expressed in the promyelocytes at a approximately two fold greater level than in the segmented neutrophils and the expression decreased as the granulocytes matured. The ratio of expression in the other leukocytes to expression in the segmented neutrophils were as follows: eosinophils/basophils approximately 7; monocytes approximately 2; lymphocytes less than 1. Expression of the HpPG gene during myeloblast differentiation was assessed by Northern blot analysis of acute myelogenous leukemia (AML) RNA samples. The expression of this gene, when compared to the levels in HL-60 cells, was approximately ten fold lower in the poorly differentiated blast cells obtained from three AML patients classified M"0". Conversely, the expression in the more differentiated blast cells obtained from 10 of 11 AML patients classified as M1 and M2 were at levels similar to the levels in HL-60 cells. The expression level found in eight lymphoid leukemias was approximately ten fold or more lower than in HL-60 cells. Gene copy number determination confirmed that the HpPG gene is present in one copy per haploid genome. Thus the HpPG gene's expression pattern denotes a single copy gene being differentially expressed during hematopoiesis with initial regulation occurring very early in this developmental process and an additional up-regulatory event occurring during granule genesis.

The isolation and identification of multiple forms of the neutrophil granule peptides from human leukemic cells.

HP-1 is a 30-residue cysteine- and arginine-rich peptide of the human neutrophil primary granule and is the most abundant human representative of the family of peptides variously called defensins and corticostatins. Peptides belonging to this family have many biological activities including the non-oxidative destruction of ingested microorganisms, the inhibition of adrenocorticotropin-stimulated synthesis of glucocorticoids, monocyte chemotaxis, the non-cytolytic inhibition of [3H]thymidine incorporation in HL-60 promyelocyte-like cells and the stimulation of nifedipine-sensitive calcium channels. Using a combination of reversed-phase and size-exclusion high performance liquid chromatography and an HP-1 radio-immunoassay, three immunoreactive peptides were detected and isolated from the promyelocyte-like cell line, HL-60, and from leukocytes of patients with chronic myelogenous and chronic lymphocytic leukemias. One of these peptides was HP-1 itself. A second was identified by gas-phase Edman microsequencing as an amino-terminally extended fragment of the HP-1 precursor which we call HP1-56. The third is likely to arise from enzymatic cleavage of the precursor at a dibasic site. Of the leukemic cells the greatest amount of HP1-56 relative to HP-1 was found in cells from a patient in myeloblastic crisis but overall the richest source of HP1-56 relative to HP-1 was found to be in fetal lung tissue. HP1-56 is difficult to detect in normal peripheral neutrophils and its presence in cells that are actively biosynthesizing primary granule components such as HL-60 may make it useful for studying the biosynthesis of granule polypeptides, their ontogeny, and possibly as a marker protein for leukemic diseases.

Genomic changes on the short arm of human chromosome 1 in breast cancer.

A total of 38 DNA sample pairs from normal and cancerous breasts were specifically examined for alterations on the short arm of chromosome 1 using probe pYNZ2. Of the pairs, 24 (63%) displayed changes at this locus including amplifications, deletions, gene rearrangements, and altered band intensities between heterozygous alleles. Due to technical considerations, this number is likely to be a conservative estimate of the frequency of alterations in this region. When data using a different probe from the short arm of chromosome 1 was added, the overall involvement of this region increased to 73%. The combined data indicate we have localized a region, likely to be less than 85 cm in size, that is involved in the development of breast cancer.

Chromosomal abnormalities in human breast cancer.

This review emphasizes cytogenetic changes and DNA analyses by Southern blot in primary breast tumors, rather than metastases, established cell lines, and pleural effusions. The data suggests that the most frequently altered chromosomes and chromosome regions are 1p, 1q, 2q, 3p, 5, 6q, 8p, 8q, 11p, 11q, 12, 13q, 14q, 16, 17p, and 17q. Changes on 8q, 11p, 11q, 13q, and 17q appear to be associated with either progression of the disease or poor prognosis. Alterations on 1p and 3p may represent early events in the development of breast cancer.

A myeloid-related sequence that localizes to human chromosome 8q21.1-22.

A myeloid-related sequence (mrs) has previously been identified that is highly expressed in selected subpopulations of myeloid leukocytes. Nucleotide sequence analysis indicates that mrs encodes what is apparently a unique 93-amino acid protein that includes an 18-amino acid leader sequence. Hybridization of an mrs cDNA probe to a Southern blot made from somatic cell hybrid DNAs shows 100% concordance with human chromosome 8, thus indicating that mrs localizes to this chromosome. In situ hybridization to metaphase chromosomes further sublocalizes mrs to bands 8q21.1-23 as 58% of the grains displayed on chromosome 8 were clustered in this region. This area encompasses the translocation breakpoint 8q22, which is rearranged in an estimated 18% of patients diagnosed with the M2 subclassification of acute nonlymphocytic leukemia (M2-ANLL). When Southern blot hybridization was performed by using somatic cell hybrid DNAs harboring either a single 8q- or a single 21q+ chromosome from two different patients with M2-ANLL, a signal was only detected in the hybrid containing the 8q-chromosome.