The clinical impact of preoperative body composition differs between male and female colorectal cancer patients.

AIM: Patient body composition is an important indicator of metabolic status and is associated with cancer progression. Because body composition varies between men and women, we aimed to examine the difference in clinical impact of preoperative body composition according to sex. METHOD: We used an integrated dataset of 559 colorectal cancer (CRC) patients. The association between preoperative body composition indices [body mass index (BMI), visceral to subcutaneous fat area ratio (VSR) and skeletal muscle index (SMI)] and patient outcome, clinicopathological factors and preoperative inflammation and nutritional status was analysed, comparing men and women. RESULTS: Preoperative low BMI and low SMI in men was significantly associated with unfavourable overall survival (OS) [BMI: hazard ratio (HR) 2.22, 95% CI 1.28-4.14, P = 0.004; SMI: HR 2.54, 95% CI 1.61-4.07, P < 0.001] and high VSR in women was significantly associated with unfavourable OS (HR 1.79, 95% CI 1.03-3.02, P = 0.040). Additionally, low SMI in men was significantly associated with deeper tumour invasion and greater distant metastasis and high VSR in women was significantly associated with advanced age, right-sided tumour, lower total lymphocyte count and lower albumin levels. Interestingly, low BMI in men was significantly associated with deeper tumour invasion, but also with favourable inflammation and nutritional status (lower C-reactive protein and higher albumin). CONCLUSION: The clinical impact of preoperative body composition differed between men and women: SMI in men and VSR in women were good prognosticators. Our findings may provide a novel insight for CRC treatment strategies.

The impact of preoperative anaemia and anaemic subtype on patient outcome in colorectal cancer.

AIM: Preoperative anaemia is associated with adverse outcomes in colorectal cancer (CRC). To clarify the reason for this we aimed to comprehensively assess the association of preoperative anaemia with tumour characteristics, host systemic inflammation and nutrition status, and perioperative blood transfusion. METHOD: We used an integrated database of 592 CRC patients. The association of preoperative anaemic subtype, calculated from haemoglobin and erythrocyte mean corpuscular volume levels, with patient outcome, preoperative serum data relating to systemic inflammation and nutrition and perioperative blood transfusion was analysed. RESULTS: Preoperative anaemia was significantly associated with poorer overall survival and relapse-free survival (RFS); in particular microcytic anaemia had a trend to poorer RFS than other forms of anaemia (P = 0.0648). In addition, preoperative anaemia was significantly correlated with right-sided tumours, greater depth of tumour invasion, use of neoadjuvant chemotherapy, poorer prognostic nutritional index and higher modified Glasgow Prognostic Score (mGPS). Microcytic anaemia in particular had a strong association with a greater depth of tumour invasion (P = 0.0072) and higher mGPS (P = 0.0058) than other causes of anaemia. Perioperative blood transfusion for CRC patients with anaemia was associated with adverse outcomes. CONCLUSIONS: Preoperative anaemia, especially microcytic anaemia, was associated with poor patient outcomes, possibly due to poor systemic inflammatory and nutritional status, and it was not improved by perioperative blood transfusion. Our data suggest that preoperative anaemia and the anaemic subtype may serve as an easily available predictor of outcome in CRC.

Minimally invasive esophagectomy may contribute to long-term respiratory function after esophagectomy for esophageal cancer.

Evidence suggests that minimally invasive esophagectomy has several advantages with regard to short-term outcomes, compared to open esophagectomy in esophageal cancer patients. However, the impact of minimally invasive esophagectomy on long-term respiratory function remains unknown. The objective of this study is to assess the association between use of the minimally invasive esophagectomy and long-term respiratory dysfunction in esophageal cancer patients after esophagectomy. This retrospective single institution study using prospectively collected data included 87 consecutive esophageal cancer patients who had undergone esophagectomy. All patients underwent a respiratory function test before, and one year after esophagectomy. Logistic regression analysis was used to compute the hazard ratio for long-term respiratory dysfunction. Minimally invasive esophagectomies were performed in 53 patients, and open esophagectomies in 34 patients. The two groups showed no significant differences in terms of postoperative complications and postoperative course. Nor were any differences observed between the two groups in terms of volume capacity (L) and forced expiratory volume 1.0 (L) before esophagectomy (P > 0.34). However, one year after esophagectomy, the decreases in volume capacity and forced expiratory volume 1.0 were significantly less in the minimally invasive esophagectomy group than in the open esophagectomy group (P = 0.04 and P = 0.007, respectively). Multivariate analyses revealed that minimally invasive esophagectomy was an independent favorable factor for maintenance of forced expiratory volume 1.0 (hazard ratio = 0.17, 95% confidence interval 0.04-0.71; P = 0.01). Minimally invasive esophagectomy may be an independent favorable factor for maintenance of long-term respiratory function in esophageal cancer patients after esophagectomy.

Monitoring sputum culture in resected esophageal cancer patients with preoperative treatment.

Pneumonia is a major cause of postesophagectomy mortality and worsens the long-term survival in resected esophageal cancer patients. Moreover, preoperative treatments such as chemotherapy or chemoradiotherapy (which have recently been applied worldwide) might affect the bacterial flora of the sputum. To investigate the association among preoperative treatments, the bacterial flora of sputum, and the clinical and pathological features in resected esophageal cancer patients, this study newly investigates the effect of preoperative treatments on the bacterial flora of sputum. We investigated the association among preoperative treatments, the bacterial flora of sputum, and clinical and pathological features in 163 resected esophageal cancer patients within a single institution. Pathogenic bacteria such as Candida (14.1%), Staphylococcus aureus (6.7%), Enterobacter cloacae (6.1%), Haemophilus parainfluenzae (4.9%), Klebisiella pneumoniae (3.7%), Methicillin-resistant Staphylococcus aureus (MRSA) (3.7%), Pseudomonas aeruginosa (2.5%), Escherichia coli (1.8%), Streptococcus pneumoniae (1.8%), and Haemophilus influenzae (1.2%) were found in the sputum. The pathogen detection rate in the present study was 34.3% (56/163). In patients with preoperative chemotherapy and chemoradiotherapy, the indigenous Neisseria and Streptococcus species were significantly decreased (P= 0.04 and P= 0.04). However, the detection rates of pathogenic bacteria were not associated with preoperative treatments (all P> 0.07). There was not a significant difference of hospital stay between the sputum-monitored patients and unmonitored patients (35.5 vs. 49.9 days; P= 0.08). Patients undergoing preoperative treatments exhibited a significant decrease of indigenous bacteria, indicating that the treatment altered the bacterial flora of their sputum. This finding needs to be confirmed in large-scale independent studies or well-designed multicenter studies.

Prognostic and clinical impact of sarcopenia in esophageal squamous cell carcinoma.

Recently, depletion of skeletal muscle mass (sarcopenia) has been linked to poor prognosis in several types of cancers, but has not been investigated in esophageal squamous cell carcinoma (ESCC). This retrospective study investigates the relationship between sarcopenia and clinical outcome in ESCC patients treated by surgical resection or definitive chemoradiation therapy (dCRT). The study was retrospectively conducted in a single academic hospital in Kumamoto, Japan, and involved 325 ESCC patients (256 surgical cases and 69 dCRT cases) treated between April 2005 and April 2011. Skeletal muscle mass was quantified by radiologic measures using standard computed tomography scans. The skeletal muscle tissue in the 325 ESCC patients was distributed as follows: mean: 47.10; median: 46.88; standard deviation (SD): 7.39; range: 31.48-71.11; interquartile range, 46.29-47.90. Skeletal muscle tissue was greater in male patients than in female patients (P < 0.0001), but was independent of other clinical and tumor features. Sarcopenia was not significantly associated with overall survival (log rank P = 0.54). Lymph node involvement significantly altered the relationship between sarcopenia and survival rate (P for interaction = 0.026). Sarcopenia significantly reduced the overall survival of patients without lymph node involvement (log rank P = 0.035), but was uncorrelated with overall survival in patients with lymph involvement (log rank, P = 0.31). The anastomosis leakage rate was significantly higher in the sarcopenia group than in the non-sarcopenia group (P = 0.032), but other surgical complications did not significantly differ between the two groups. Sarcopenia in ESCC patients without lymph node involvement is associated with poor prognosis, indicating sarcopenia as a potential biomarker for identifying patients likely to experience an inferior outcome. Moreover, sarcopenia was associated with anastomosis leakage but no other short-term surgical outcome.

Statins inhibit tumor progression via an enhancer of zeste homolog 2-mediated epigenetic alteration in colorectal cancer.

While statin intake has been proven to reduce the risk of colorectal cancer (CRC), the mechanism of antitumor effects and clinical significance in survival benefits remain unclear. Statin-induced antiproliferative effects and its underlying mechanism were examined using six CRC cell lines. Statins except pravastatin showed antiproliferative effects (simvastatin ≥ fluvastatin > atorvastatin) even though both of simvastatin and pravastatin could activate mevalonate pathways, suggesting the statin-mediated antiproliferative effects depended on non-mevalonate pathway. Indeed, statin induced p27(KIP1) expression by downregulation of histone methyltransferase enhancer of zeste homolog 2 (EZH2), which acts as an epigenetic gene silencer. Additionally, the use of simvastatin plus classII histone deacetylase (HDAC) inhibitor (MC1568) induced further overexpression of p27(KIP1) by inhibiting HDAC5 induction originated from downregulated EZH2 in CRC cells and synergistically led to considerable antiproliferative effects. In the clinical setting, Statin intake (except pravastatin) displayed the downregulated EZH2 expression and inversely upregulated p27(KIP1) expression in the resected CRC by immunohistochemical staining and resulted in the significantly better prognoses both in overall survival (p = 0.02) and disease free survival (p < 0.01) compared to patients without statin intake. Statins may inhibit tumor progression via an EZH2-mediated epigenetic alteration, which results in survival benefits after resected CRC. Furthermore, statin plus classII HDAC inhibitor could be a novel anticancer therapy by their synergistic effects in CRC.

Possible involvement of calcium-calmodulin pathways in the positive chronotropic response to angiotensin II on the canine cardiac sympathetic ganglia.

We investigated the ganglionic effects of angiotensin II (Ang II) and the signal transduction involved in the cardiac sympathetic ganglia by the direct administration of agents to the ganglia through the right subclavian artery and monitoring the heart rate as an indicator of the ganglionic function in pithed dogs. Ang II given i.a. caused increases in the heart rate, which was inhibited by the treatment with the AT1-receptor antagonist forasartan, but not by the AT2-receptor antagonist PD-123319. The stimulation by Ang II, but not by acetylcholine, was inhibited after treatment with an inhibitor of phospholipase C, U-73122; a cell-permeant modulator of the Ins(1,4,5)P3 receptors, 2-aminoethoxydiphenyl borate; an intracellular calcium and calcium-associated protein kinase inhibitor, HA-1077; calmodulin (CaM) inhibitor, W-7; Ca2+/CaM-dependent protein kinase II inhibitor, KN-93; a selective protein kinase C inhibitor, calphostin C; and Na+H+ exchange inhibitor, dimethylamiloride. These results suggest that Ang II stimulates the ganglionic transmission at postsynaptic sites via the activation of AT1 receptor coupled to either activation of phospholipase C, phosphoinositide hydrolysis and subsequent increase in intracellular Ca2+ and activation of protein kinase C and Ca2+/CaM kinase II, although this ganglionic stimulation seems to involve, at least in part, the protein kinases-dependent increase of amiloride-sensitive Na+ inflow.

Involvement of PLAGL2 in activation of iron deficient- and hypoxia-induced gene expression in mouse cell lines.

We searched iron-deficient inducible cDNA, using subtraction cloning and mRNA from desferrioxamine-treated mouse macrophage Raw264.7 cells. We identified a pleomorphic adenoma gene like 2 (PLAGL2), one of PLAG superfamily proteins exhibiting antiproliferative properties on tumor cells. Mouse PLAGL2 consists of 496 amino acids with seven C2H2 zinc-fingers. PLAGL2 mRNA was induced in RAW264.7 cells, mouse erythroleukemia cells and Balb/c 3T3 cells when they were treated with desferrioxamine. Hypoxia also increased PLAGL2 mRNA. Expression of PLAGL2 in COS-7 cells led to nuclear localization. PLAGL2 had potential binding ability to GC-rich oligonucleotide and activated transcription of a gene with the binding sequence in transient reporter assay, a finding consistent with a case seen in a PLAGL2 homolog, ZAC-1. Transient co-transfection of PLAGL2 or ZAC1 cDNA and a reporter containing a lactate dehydrogenase A (LDHA) promoter carrying the hypoxia inducible factor-1 responsive element led to an increase in the basal transcription in Balb/c 3T3 and HepG2 cells. Activation in transcription from the LDHA promoter increased by desferrioxamine treatment or hypoxia was further enhanced when PLAGL2 was expressed. We propose that PLAGL2 is involved in the cell cycle arrest and apoptosis of tumor cells by regulating iron depletion- or hypoxia-inducible gene expression.

Upregulation of immunoreactive angiotensin II release and angiotensinogen mRNA expression by high-frequency preganglionic stimulation at the canine cardiac sympathetic ganglia.

The possible involvement of the local angiotensin system in ganglionic functions was investigated in the canine cardiac sympathetic ganglia. Positive chronotropic responses to preganglionic stellate stimulation at high frequencies, after intravenous administration of pentolinium plus atropine, were inhibited by the nonpeptide angiotensin AT(1) receptor antagonist forasartan or the angiotensin I-converting enzyme inhibitor captopril, whereas the rate increases elicited by the postganglionic stellate stimulation and norepinephrine given intravenously failed to be inhibited by these antagonists. The levels of endogenous immunoreactive angiotensin II, as determined by radioimmunoassay in the incubation medium of the stellate and inferior cervical ganglia, were increased after the high-frequency preganglionic stimulation of the isolated ganglia. The increment of the peptide was also antagonized by the pretreatment with captopril but not by a chymase inhibitor, chymostatin. The expression of angiotensinogen mRNA was observed in the stellate ganglion, adrenal, liver, and lung but not in the ovary and spleen. The expression of the mRNA in the stellate and inferior cervical ganglia increased after high-frequency preganglionic stimulation of the in vivo dogs for a period of 1 hour. These results indicate that an intrinsic angiotensin I-converting enzyme-dependent angiotensin system exists in the cardiac sympathetic ganglia, which is activated by high-frequency preganglionic stimulation.

Analysis of the candidate genes responsible for non-syndromic cleft lip and palate in Japanese people.

In order to assess the association of alleles for candidate genes with non-syndromic cleft lip and palate, DNA samples from 43 Japanese patients were compared with those from 73 control subjects with respect to the genes encoding transforming growth factor alpha (TGFalpha), TGFbeta and gamma-aminobutyric acid type A receptor beta3 (GABRB3). The restriction fragment length polymorphisms of the 3'-non-coding region of the TGFalpha gene K-primer region were observed after digestion with NcoI and HinfI. Allele 4 was the most common among cases of cleft lip with or without cleft palate, whereas allele 2 was the most common among controls. A significant difference was found in this region between groups with cleft lip (with or without cleft palate) and controls (chi2=10.190; P=0.017). Three alleles of the TGFbeta2 gene were tested, and allele 2 was the most common in both cases and controls. The proportion of allele 2 in the case group was greater than that in the control group, showing a significant difference between cases of cleft lip (with or without cleft palate) and controls (chi(2)=19.208; P<0.0001). No significant differences in variants of TGFbeta3 or GABRB3 between case and control populations were observed. Thus it is concluded that TGF genes play a role in craniofacial development, and that alleles of TGFalpha or/and TGFbeta2 are associated with cleft lip and cleft palate in Japanese populations.

ELISA for urinary trehalase with monoclonal antibodies: a technique for assessment of renal tubular damage.

BACKGROUND: alpha,alpha-Trehalase, located on renal proximal tubules, is a glycoprotein that hydrolyses alpha,alpha-trehalose to two glucose molecules. Urinary trehalase reflects damage to renal proximal tubules, but its activity has not been measured routinely because measurement of catalytic activity is rather complicated and because conventional assays for enzyme activity might not reflect all of the trehalase protein because of enzyme inactivation in urinary samples. METHODS: We established novel monoclonal antibodies for human trehalase and a sandwich ELISA for quantification of urinary trehalase. We determined the urinary trehalase protein concentration with this ELISA and trehalase catalytic activity, and the results of these two methods were compared. RESULTS: The ELISA system was more sensitive than the detection of enzyme activity and could detect a subtle difference in the amount of trehalase present in renal diseases. The within- and between-assay CVs in the ELISA were 6.7-7.6% and 6.2-8.2%, respectively. Highly significant increases in both the quantity and activity were seen in patients with nephrotic syndrome (acute phase), Lowe syndrome, and Dent disease. The quantities were 70- to 200-fold greater, whereas enzyme activities were, at most, 10-fold higher than those of control subjects. In the detection of small amounts of trehalase in patients with chronic glomerulonephritis and renal anomalies, quantities were better than enzyme activities. CONCLUSIONS: We have established an ELISA system for quantification of urinary trehalase that uses novel monoclonal antibodies. Our ELISA system is simpler and more sensitive than a conventional activity assay and reflects trehalase protein. This ELISA can be a useful as a common tool for clinical assessment of renal proximal tubular damage.

Morphological change, loss of deltapsi(m) and activation of caspases upon apoptosis of colorectal adenocarcinoma induced by 5-FU.

Apoptosis is clearly distinguished from necrosis, morphologically and chemically. Morphologically, apoptosis is characterized by a condensed nucleus and the disappearance of microvilli without disruption of the cytoplasm. In this report, we demonstrate that 5-fluorouracil (5-FU)-induced early apoptotic cells are characterized by (i) ultracondensed mitochondria, (ii) no change in the microvilli or nucleus, (iii) a high mitochondrial transmembrane potential (Deltapsi(m)), and (iv) being annexin V(negative). The early apoptotic cells also show the active forms of caspase 8 and caspase 9. They rapidly lose Deltapsi(m) after further incubation. Therefore, we conclude that the ultracondensation of mitochondria precedes the loss of Deltapsi(m) and the exposure of phosphatidylserine to the outer leaflet of the cell membrane.

Cloning and expression of the M5 RNA segment encoding outer capsid VP5 of epizootic hemorrhagic disease virus Japan serotype 2, Ibaraki virus.

The complete nucleotide sequence of a cDNA clone representing the M5 RNA segment of epizootic hemorrhagic disease virus Japan serotype 2 (EHDV-2), Ibaraki virus, was determined. The M5 segment is 1641 base pairs long with the single open reading frame which predicts a polypeptide of 527 amino acids. The comparison of the amino acid sequence of the VP5 with those of EHDV-1, bluetongue virus serotype 10, and African horse sickness virus serotype 4 revealed that the protein shared 67%, 57% and 42% homologies, respectively. In addition, the VP5 protein was expressed in insect cells by recombinant baculovirus, which could be recognized by the mouse anti-EHDV-2 sera at a position of the expected 59 kDa on immunoblot analysis.

Activated Rac1 selectively up-regulates the expression of integrin alpha6beta4 and induces cell adhesion and membrane ruffles of nonadherent colon cancer Colo201 cells.

Functions of small GTPases in integrin expression were investigated when the interaction of nonadherent human colon carcinoma 201 cells with the extracellular matrix (ECM) was examined. By transfection of the constitutively active form of a small GTPase Rac1, Rac V12, adhesion of cells to the ECM increased with concomitant cell spreading and formation of membrane ruffles. Activated Cdc42 and Cdc42 V12, but not wild-type Rac1, Cdc42, or RhoA, also induced the adhesion and spreading of Colo201 cells. This adhesion is integrin beta4 dependent since an antibody for integrin beta4 inhibited the RacV12-dependent cell adhesion and numbers of adhesive cells on laminin-coated plates exceeded those on collagen- and fibronectin-coated plates. By immunofluorescence, in addition to clustering of integrin molecules, expression of integrin alpha6beta4 on the cell surface of Rac V12- and Cdc42 V12-expressing cells was selectively up-regulated without an increase in biosynthesis of alpha6beta4 integrin. Treatment of Rac V12-expressing cells with wortmannin or LY294002, specific inhibitors of phosphoinositide 3-OH kinase, decreased the up-regulated alpha6beta4 and cell adhesion. In light of this evidence, we propose that the regulation of integrin alpha6beta4 expression induced by Rac1 and Cdc42 may play an important role in cell adhesion and tumorigenesis of colon carcinoma cells.

Apoptosis of colorectal adenocarcinoma induced by 5-FU and/or IFN-gamma through caspase 3 and caspase 8.

The cytotoxic effect of 5-FU has been shown by the induction of apoptosis in cancer cells, and reported to be enhanced by IFN-gamma. We examined the role of caspases on the apoptosis induction of 5-FU and IFN-gamma using a colorectal adenocarcinoma cell line. The activities of caspase 3 and caspase 8 increased when apoptosis was induced by 5-FU and/or IFN-gamma. Moreover, all apoptotic cells showed high caspase 3 activity in these conditions. Although the inhibitors of caspase 3 and caspase 8 inhibit apoptosis, anti-Fas ligand antibody does not affect the apoptosis induced by 5-FU. Thus, caspase 3 and caspase 8 play crucial roles in apoptosis induced by 5-FU and/or IFN-gamma, regardless of the Fas-Fas ligand system.

A crucial role of caspase 3 and caspase 8 in paclitaxel-induced apoptosis.

The anticancer drug paclitaxel is well known as an inhibitor of microtubule depolymerization, resulting in mitosis arrest. We investigated the mechanism underlying antitumor effects of paclitaxel on the lung adenocarcinoma cell line LC-2-AD. Less than 10 microg/ml paclitaxel induced mitosis arrest upon LC-2-AD, followed by apoptosis, but more than 30 microg/ml paclitaxel induced apoptosis without mitosis arrest. LC-2-AD with less than 1 microg/ml paclitaxel showed a loss of mitochondrial transmembrane potential (deltapsim), which correlated with antitumor effects. However, LC-2-AD with more than 10 microg/ml paclitaxel showed slight changes in the loss of deltapsim in spite of its ability to induce apoptosis significantly. The cleavage of caspase 3, caspase 8, and DFF45/ICAD was also observed in paclitaxel-induced apoptosis, and the inhibitor of caspase 3 and caspase 8 inhibited both antitumor effects and apoptosis induced by paclitaxel. These results suggest that activation of caspase 3 and caspase 8 plays a crucial role in paclitaxel-induced apoptosis under any concentrations of paclitaxel.

MAP kinase-independent induction of proto-oncogene c-fos mRNA by hemin in human cells.

Treatment of HeLa cells or human skin fibroblast cells with hemin led to a time- and dose-dependent rapid induction of c-fos mRNA. This induction was absent in the cells treated with actinomycin D, indicating that the c-fos induction by hemin occurs at the level of transcription. Metalloporphyrins, including zinc-, cobalt-, and tin-protoporphyrin, ferric ion, and protoporphyrin also induced c-fos mRNA. Transient reporter assay with the reporter constructs of the human c-fos gene promoter up to -404 bp connected to the luciferase gene showed high activity but no induction by hemin, suggesting that cis-acting elements, including the serum response element located about -310 bp upstream of the human c-fos gene promoter, may not contribute to the heme-dependent induction. With in-gel assay of protein kinases, the activity of the mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase 12 or p38 MAP kinase in hemin-treated HeLa cells was not stimulated. Stimulation of c-Jun N-terminal kinase by hemin was nil. Furthermore, PD58059 and SB203580, inhibitors for MAP kinases, did not affect the hemin-dependent c-fos induction. Of the inhibitors for protein kinases so far tested, KN-62, a specific inhibitor for calmodulin-dependent protein kinase II (CaMK II), inhibited the induction of c-fos mRNA by hemin. Phosphorylation of CaMK II in hemin-treated cells increased. With gel mobility assay, the DNA AP-1 binding activity transiently increased when treating HeLa cells with hemin. Therefore, induction of c-fos led to an activation of AP-1 in the presence of hemin. We suggest that calmodulin-dependent protein kinase II rather than the MAP kinase family regulates the induction of the human c-fos gene expression by hemin.

Structure and transcriptional regulation of the mouse ferrochelatase gene.

Ferrochelatase (EC.4.99.1.1), the final step in the biosynthesis of heme, is widely expressed in various tissues and is induced in erythroid cells. We determined the structure of the mouse ferrochelatase gene after isolation and characterization of lambda phage clones mapping discrete regions of the cDNA. The gene spans about 25 kb and consists of 11 exons. The exon/intron boundary sequences conform to consensus acceptor (GTn)/donor (nAG) sequences, and exons in the gene encode functional protein domains. The promoter region contains multiple Sp1 sites, a CACCC box and GATA-1 binding sites. Function analysis of the promoter by transient transfection assay demonstrated that one Sp1 binding site located at -37/-32 is essential for basic expression of the ferrochelatase gene in both mouse erythroleukemia (MEL) and non-erythroid EL4 cells. In addition, the region (-66/-51) containing a CACCC box and the neighboring GC box partly contributes to the inducible activity of the reporter in MEL cells upon induction with dimethylsulfoxide. It appears that at least two promoter regions of the mouse ferrochelatase gene function in basic and inducible expression.

Molecular characterization of a newly identified heme-binding protein induced during differentiation of urine erythroleukemia cells.

A heme-binding protein with a molecular mass of 22 kDa, termed p22 HBP, was purified from mouse liver cytosol, using blue Sepharose CL-6B. We identified a cDNA encoding p22 HBP, and sequence analysis revealed that p22 HBP comprises 190 amino acid residues (Mr 21,063) and has no homology to any other known heme-binding protein. The p22 HBP mRNA (approximately 1.0 kilobases) is ubiquitously expressed in various tissues and is extremely abundant in the liver. cDNA allows for expression of active p22 HBP, with a high affinity for 55Fe-hemin, with a Kd of 26 +/-1.8 nM. The Bmax of hemin binding to p22 HBP was 0.55 +/- 0.021 mol/mol of protein, a value consistent with one heme molecule binding per molecule of protein. The order of potency of different ligands to compete against 55Fe-hemin binding to p22 HBP was hemin = protoporphyrin IX > coproporphyrin III > bilirubin > palmitic acid > all-trans-retinoic acid. Treatment of mouse erythroleukemia (MEL) cells with dimethyl sulfoxide or hemin resulted in an increase in p22 HBP mRNA. The immunoblot analysis showed that p22 HBP increased with time in dimethyl sulfoxide- and hemin-induced MEL cells. Conversely, transfer of antisense oligonucleotides to p22 HBP cDNA resulted in a decrease of p22 HBP in dimethyl sulfoxide-treated MEL cells, and the heme content in these cells decreased to 66-71% of sense oligonucleotides-transferred cells. Thus, this newly identified heme-binding protein, p22 HBP, may be involved in heme utilization for hemoprotein synthesis and even be coupled to hemoglobin synthesis during erythroid differentiation.