Nader G. Abraham

Overexpression of the Heme Oxygenase Gene in Renal Cell Carcinoma

Heme oxygenase (HO) activity has been implicated in the regulation of renal function and cell growth in normal and disease states. Expression of HO genes has been shown to regulate important hemoprotein(s) such as cytochrome P450. In the present study, HO activity was measured in samples of human adenocarcinoma, jux-tatumor, and normal renal tissues. The samples were histologically examined to verify the malignant and normal nature. HO activity was 4-fold higher in the adenocarcinoma than in either normal or juxtatumor tissues. We designed a reverse transcriptase-polymerase chain reaction (RT-PCR) method to assess the presence of HO-1 and HO-2 mRNA in biopsy samples of various human renal tissues. Total RNA from renal samples was reverse transcribed and amplified simultaneously by PCR using specific primers for HO-1 and HO-2. Results show that both HO-1 and HO-2 mRNAs were expressed in all renal tissues examined and that HO-1 appeared to be amplified more than HO-2. Northern blot analysis revealed that HO-1 mRNA was elevated by several-fold in adenocarcinoma compared with juxtatumor or normal tissues. In contrast, no differences in HO-2 mRNA levels were observed using either RT-PCR or Northern blot. Cytochrome P450 arachidonic acid epoxygenase and ω-hydroxylase activities were markedly reduced in the tumor tissues, whereas, in the juxtatumor tissue, cytochrome P450 ω-hydroxylase activity was significantly increased. Northern blot analysis using cytochrome P450 cDNA probe 4A2 cDNA for the ω-hydroxylase gene family revealed that mRNA levels for ω-hydroxylase transcripts were significantly decreased in the adenocarcinoma compared with juxtatumor. The decrease in cytochrome P450 4All mRNA levels correlated with a decrease in the arachidonic acid ω-hydroxylation metabolite, 20-HETE. The production of 20-HETE was significantly higher in juxtatumor in agreement with ω-hydroxylase mRNA. Higher levels of HO-1 may be a contributing factor for the undetectable levels of cytochrome P450 arachidonic acid metabolites, 20-HETE, in the adenocarcinoma. Our results suggest that increased generation of mitogenic activities by ω-hydroxylase and 20-HETE in the juxtatumor may be a contributing factor in the development and growth of neoplastic tissues, and the induction of HO in the tumor tissue may be an attempt to limit oxidative injury caused by the cytochrome P450 metabolites and other oxidative stress.

Tin-Mediated Heme Oxygenase Gene Activation and Cytochrome P450 Arachidonate Hydroxylase Inhibition in Spontaneously Hypertensive Rats

The effect of SnCl2 on the transcription of the heme oxygenase gene in spontaneously hypertensive rats was examined using cDNA for the rat heme oxygenase (HO-1). An increase in renal HO-1 mRNA levels was observed in response to SnCl2 treatment. Quantitative evaluation by scanning densitometry demonstrated a maximal increase in HO-1 mRNA 24-fold over control at 8 hours after SnCl2 administration. Nuclear runoff assay using isolated renal nuclei from SnCl2-treated rats revealed an active HO-1 gene transcription. Transcription of HO-1 in rat kidney was greatly increased within 3 hours of administration of SnCl2, as evidenced by the level of [α32P]UTP incorporation into nuclear RNA. As a consequence of activation of the HO-1 gene transcription, renal enzyme activity increased eightfold at 16 hours after SnCl2, and reached maximal activity of 16-fold over control at 32 hours after injection. No significant change in cytochrome P450 fatty acid ω-hydroxylase (P450 4A) mRNA was observed after SnCl2 administration. Cytochrome P450-arachidonic acid ω/ω-1 hydroxylase(s) activity (formation of 20− and 19-HETE) was significantly reduced 24 hours after SnCl2 administration and remained lower than the control level 48 and 72 hours after injection. In addition, blood pressure was reduced from 151 ± 2.5 mm Hg to 133 ± 2.3 mm Hg after 48 hours of SnCl2 treatment. The reduction in blood pressure preceded natriuresis. It is concluded that SnCl2 induces activation of the HO-1 gene, which is followed by elevation in enzyme activity and a decrease in cytochrome P450-arachidonic acid ω-hydroxylase activity. These biochemical changes bring about a selective decrease in the synthesis of 19-HETE and 20-HETE, arachidonate metabolites with prohypertensive properties, and are associated with blood pressure reduction to normal levels. It is suggested that manipulation of heme oxygenase and cytochrome P450-arachidonic acid ω-hydroxylase expressions may be of therapeutic importance in regulating blood pressure.

Physiologic Role of Heme and Cytochrome P-450 in Hematopoietic Cells:

Heme (ferroprotoporphyrin IX) is a ubiquitous molecule that serves as the prosthetic group of a variety of important hemoproteins that are essential for hemopoietic processes. Heme is involved in oxygen transport as the prosthetic group of hemoglobin, in prostaglandin synthesis as the prosthetic group of cyclooxygenase, in the enzymatic decomposition of H2O2 as the prosthetic group of catalase and peroxidase, and in the inactivation of oxygen molecules, as the prosthetic group of mitochondrial and microsomal cytochrome P-450. The latter refers to a family of isozymes for which heme serves as the prosthetic group that oxidizes a wide variety of structurally unrelated compounds, inactivates leukotrienes (leukotriene B4), and metabolizes arachidonic acid (AA) to bioactive metabolites, some of which are involved in the signal transduction process for hematopoietic growth factors. However, the effect of heme on erythropoiesis appears to be distinct from its direct involvement as a prosthetic group. The expression of specific heme metabolic enzymes determines the level of cellular heme that is necessary for proper erythropoiesis. This concept is supported by evidence that hereditary or experimental alterations in enzymatic or biosynthetic events are often accompanied by a disturbance in heme levels, and that treatment with drugs or inhibitors of heme synthesis may affect progenitor cells, resulting in altered growth and differentiation (1). It is becoming increasingly evident that the role of heme metabolic enzymes in the regulation of hematopoiesis has a dual nature, since metabolic enzymes appear to participate in the implementation of both stimulation and suppression of erythropoiesis (1, 2). Enhancement of erythropoiesis obtained with the growth factors interleukin 3 and erythropoietin (Epo) also results in increased levels of δ-aminolevulinic acid synthase (ALAS) and porphobilinogen deaminase (PBGD), the proposed rate-limiting enzymes in the heme biosynthetic pathway.

Cytochrome P450 Arachidonic Acid ω‐Hydroxylation in the Proximal Tubule of the Rat Kidneya

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) is a major cytochrome P450-dependent arachidonate metabolite in the rat kidney. In the present study we characterized the formation of 20-HETE in the proximal tubule, the nephron segment with the highest concentration of cytochrome P450 activities, including P450 arachidonic acid metabolism. Freshly isolated tubules showed a basal formation of 20-HETE, implying that it is an endogenous constituent of the proximal tubule. Conversion of exogenous arachidonic acid to 20-HETE in proximal tubule homogenates was enzymatic and NADPH-dependent (i.e., 0 and 65.5 +/- 1.1 pmol/mg/min in the absence and presence of NADPH, respectively). That its formation was not affected by indomethacin but inhibited following preincubation with 17-ODYA (17-octadecynoic acid) and 7-ER (7-ethoxyresorufin) suggested that a P450 monooxygenase activity was involved in its synthesis. This was further strengthened by the demonstration that antibody raised against the rat cytochrome P450 4A1, a major fatty acid omega-hydroxylase isozyme, inhibited 20-HETE formation, suggesting the involvement of a P450 4A1 or P450 4A1-like activity in this reaction. Pretreatment of rats with clofibrate and dexamethasone, inducers of the P450 4A gene family, yielded a twofold increase in the proximal tubular synthesis of 20-HETE as well as an increase in P450 4A1 mRNA. These results, together with previous demonstrations that 20-HETE vasoconstricts isolated blood vessels, namely, renal microvessels, and affects tubular ion transport, suggest a role for 20-HETE in the regulation of renal vascular tone and transport functions and further stress the importance of understanding the regulation of 20-HETE synthesis in the kidney.

Diminished heme oxygenase potentiates cell death: Pyrrolidinedithiocarbamate mediates oxidative stress

Pyrrolidinedithiocarbamate (PDTC) is a metal-chelating compound that exerts both pro-oxidant and antioxidant effects and is widely used as an antitumor and anti-inflammatory agent. Heme oxygenase-1 (HO-1) is a redox-sensitive-inducible protein that provides efficient cytoprotection against oxidative stress. Because it has been reported that several angiogenic stimulating factors upregulating HO-1 in endothelial cells cause a significant increase in angiogenesis, we investigated the effect of PDTC on cell proliferation and angiogenesis and the effect of overexpression and underexpression of HO-1. The evaluation of PDTC (20 or 50 μM) in endothelial cells resulted in significant increase in HO-1 mRNA and protein (P < 0.001), but a decrease in cell proliferation. Pretreatment of endothelial cells with SnCl2 (10 μM), an inducer of HO-1 attenuated the PDTC-mediated decrease in cell proliferation (P < 0.05). In contrast, pretreatment with SnMP, an inhibitor of HO activity, magnified the inhibiting effect of PDTC on cell proliferation. Upregulation of HO-1 gene expression by retrovirus-mediated delivery of the human HO-1 gene also attenuated the PDTC-induced decrease in cell proliferation. Underexpression of HO-1, by delivery of the human HO-1 in antisense orientation, enhanced the PDTC-mediated decrease in cell proliferation. The decrease, by PDTC, in proliferation of cells underexpressing HO-1 is related to an increase in O− 2 production. Collectively, these results demonstrate that upregulation of HO-1 was able to attenuate the PDTC-mediated cell proliferation, but was unable to reverse the high concentration of PDTC-induced decrease in angiogenesis.

Effect of Blood Substitute, Recombinant Hemoglobin, on in vivo Hematopoietic Recovery from AZT Toxicity

We determined the in vivo ability of infused human recombinant hemoglobin 1.1 (hr-Hb) and erythropoietin to rescue the hematopoietic activity from the suppressive effects of AZT in normal and in a murine model of AIDS (MAIDS) mice. Mice were fed with AZT for 8 weeks with or without treatment in the last 4 weeks by administering various concentrations of hr-Hb and/or erythropoietin (Epo). Blood parameters, body weight (BW) and erythroid burst-forming units (BFU-E) for all mice were determined. AZT-treated normal and MAIDS mice showed a significant decrease in hematocrit (64 and 78.1%), hemoglobin (27.2 and 45.5%), BW (17.5 and 35.5%), number of white (66.9 and 42.1%) and red blood cells (65.5 and 38%), and the number of BFU-E (73 and 59%), whereas the AZT-treated normal and MAIDS mice that received hr-Hb (5 mg/kg BW/day) and/or Epo (2 U/mouse/day) showed significant alleviation of AZT cytotoxicity. This was evident by the recovery in all blood indices examined, the number of BFU-E and the BW of mice treated. BFU-E recovery in MAIDS (97%) was greater than that in normal mice (63%) as compared to their controls. hr-Hb produced a similar response as the combination, however recovery was slightly better with the latter in some hematological parameters. Higher concentrations of hr-Hb (10-15 mg) did not result in a more significant increase in most blood indices. Our results indicate that infusion with hr-Hb can alleviate AZT toxicity in normal and in immunodeficient mice, and that hr-Hb may be clinically useful in preventing severe bone marrow depression brought about by various drugs or agents such as AZT.

Quantitative measurement of heme oxygenase‐1 in the human renal adenocarcinoma

Heme oxygenase (HO‐1) is the rate‐limiting enzyme in heme catabolism. HO‐1, a stress protein, has been suggested to be involved in defense mechanisms against agents that may induce oxidative stress. It has been proposed that renal HO gene expression regulates important hemoprotein(s) such as cytochrome P450 and may be essential to maintain homeostasis in the kidney. Because accurate assessment of HO‐1 mRNA in normal and disease states in kidney were not available due to the limited number of cells, we developed a system to quantitate human HO‐1 mRNA in samples limited in cell number and/or mRNA copies. Total RNA from human kidney was used to establish this technique; it was reverse‐transcribed and then amplified by polymerase chain reaction (PCR) in a tube also containing an internal standard obtained by deleting 50 bp from the original human HO‐1 gene. This allowed us to use the same primers for both the sample and internal standard. After amplification, templates were resolved by acrylamide gel electrophoresis and quantitated either by densitometry or radioactivity counted from the bands excised from the gel. When the internal standard is present in the reaction mixture, the ratio of amplified sample vs. the standard template is proportional to the amount of sample RNA, and it is therefore possible to calculate the number of specific mRNA molecules. We have used this approach to quantitate the number of HO‐1 mRNA molecules in adenocarcinoma cells. Results show that reverse transcription (RT)/PCR methods were able to determine the number of HO‐1 mRNA copies in biopsy samples of human adenocarcinoma cells.

Comparative Pharmacology of Zinc Mesoporphyrin and Tin Mesoporphyrin: Toxic Actions of Zinc Mesoporphyrin on Hematopoiesis and Progenitor Cell Mobilization

The effects of two synthetic heme analogues, zinc mesoporphyrin (ZnMP) and tin mesoporphyrin (SnMP), on in vivo hematopoietic progenitor cell mobilization and in vitro hematopoiesis were examined in rabbit bone marrow. Rabbits received granulocyte colony-stimulating factor (rhG-CSF) for 7 days in order to mobilize increased numbers of erythroid (BFU-E) and myeloid (CFU-GM) progenitors in peripheral blood. Concurrent treatment of rhG-CSF-treated rabbits with ZnMP reduced mobilization of the numbers of BFU-E (76% inhibition, p < 0.0001) and CFU-GM (70% inhibition, p < 0.005) in peripheral blood. In contrast, SnMP administered at the same concentration had no significant suppressive effect on BFU-E and CFU-GM recruitment. Both metalloporphyrins inhibited bone marrow heme oxygenase activity equally in vivo, thus indicating that both compounds enter bone marrow cells. Direct in vitro addition of ZnMP to normal rabbit bone marrow cultures suppressed BFU-E and CFU-GM growth, whereas SnMP had no such effect. These results confirm, in an in vivo system, our earlier in vitro studies and demonstrate that, at the concentrations studied, ZnMP, in contrast to SnMP, displays toxicity for hematopoietic growth and progenitor cell production.

Specific inhibition of c-fos proto-oncogene expression by triple-helix-forming oligonucleotides.

The promoter region of the c‐fos oncogene 5′ flanking sequence contains enhancer elements crucial for binding nuclear factors that regulate transcription following cell proliferation and differentiation. Single‐stranded deoxyoligonucleotides were chosen for modulation of c‐fos protooncogene expression because of their high‐affinity binding to specific nucleotide sequences. We designed two oligonucleotides that form a triple‐helix complex on the retinoblastoma gene product‐responsible element of the c‐fos oncogene.

Characterization of a 142-bp fragment of the murine c-fos oncogene promoter upstream of the SIF-binding element.

We previously reported that in transformed mouse sarcoma cells of spontaneous origin and in revertants transfected with a fos-cat fusion, the 600-bp c-fos promoter region provides chloramphenicol acetyltransferase activity. In the present study, we investigated the binding of transcriptional factor protein(s) to a region (-503 to -361) upstream of the sis (platelet-derived growth factor)-inducible factor (SIF)-binding element. Gel electrophoresis retardation (GER) assay clearly demonstrated the appearance of strong binding activity to a newly described fragment in the 142-bp region studied. Further analysis using synthetic oligodeoxyribonucleotides and GER defined a binding region of 30 bp (AvaI-AvaII) from -503 to -472 that partially overlaps with a region known to bind fos promoter binding site 2 (FBS2). DNase I footprint analysis discovered a novel sequence in the upstream region of the c-fos promoter to which protein(s) in nuclear extracts from various mouse and human cells bind. This factor(s) is not identical to most known transcriptional factors present in the promoter region of nuclear oncogenes. A proximal part of this fragment is very conservative and contains several AP-2-like-binding sites.