Uroporphyrinogen III synthase. An alternative promoter controls erythroid-specific expression in the murine gene.

Uroporphyrinogen III synthase (URO-synthase, EC 4.2.1.75) is the fourth enzyme of the heme biosynthetic pathway and is the defective enzyme in congenital erythropoietic porphyria. To investigate the erythroid-specific expression of murine URO-synthase, the cDNA and approximately 24-kilobase genomic sequences were isolated and characterized. Three alternative transcripts were identified containing different 5'-untranslated regions (5'-UTRs), but identical coding exons 2B through 10. Transcripts with 5'-UTR exon 1A alone or fused to exon 1B were ubiquitously expressed (housekeeping), whereas transcripts with 5'-UTR exon 2A were only present in erythroid cells (erythroid-specific). Analysis of the TATA-less housekeeping promoter upstream of exon 1A revealed binding sites for ubiquitously expressed transcription factors Sp1, NF1, AP1, Oct1, and NRF2. The TATA-less erythroid-specific promoter upstream of exon 2A had nine putative GATA1 erythroid enhancer binding sites. Luciferase promoter/reporter constructs transfected into NIH 3T3 and mouse erythroleukemia cells indicated that the housekeeping promoter was active in both cell lines, while the erythroid promoter was active only in erythroid cells. Site-specific mutagenesis of the first GATA1 binding site markedly reduced luciferase activity in K562 cells (<5% of wild type). Thus, housekeeping and erythroid-specific transcripts are expressed from alternative promoters of a single mouse URO-synthase gene.

Congenital erythropoietic porphyria successfully treated by allogeneic bone marrow transplantation.

The long-term biochemical and clinical effectiveness of allogenic bone marrow transplantation (BMT) was shown in a severely affected, transfusion-dependent 18-month-old female with congenital erythropoietic porphyria (CEP), an autosomal recessive inborn error of heme biosynthesis resulting from mutations in the uroporphyrinogen III synthase (URO-synthase) gene. Three years post-BMT, the recipient had normal hemoglobin, markedly reduced urinary porphyrin excretion, and no cutaneous lesions with unlimited exposure to sunlight. The patient was homoallelic for a novel URO-synthase missense mutation, G188R, that expressed less than 5% of mean normal activity in Escherichia coli, consistent with her transfusion dependency. Because the clinical severity of CEP is highly variable, ranging from nonimmune hydrops fetalis to milder, later onset forms with only cutaneous lesions, the importance of genotyping newly diagnosed infants to select severely affected patients for BMT is emphasized. In addition, the long-term effectiveness of BMT in this patient provides the rationale for future hematopoietic stem cell gene therapy in severely affected patients with CEP.

Familial porphyria cutanea tarda: characterization of seven novel uroporphyrinogen decarboxylase mutations and frequency of common hemochromatosis alleles.

Familial porphyria cutanea tarda (f-PCT) results from the half-normal activity of uroporphyrinogen decarboxylase (URO-D). Heterozygotes for this autosomal dominant trait are predisposed to photosensitive cutaneous lesions by various ecogenic factors, including iron overload and alcohol abuse. The 3.6-kb URO-D gene was completely sequenced, and a long-range PCR method was developed to amplify the entire gene for mutation analysis. Four missense mutations (M165R, L195F, N304K, and R332H), a microinsertion (g10insA), a deletion (g645Delta1053), and a novel exonic splicing defect (E314E) were identified. Expression of the L195F, N304K, and R332H polypeptides revealed significant residual activity, whereas reverse transcription-PCR and sequencing demonstrated that the E314E lesion caused abnormal splicing and exon 9 skipping. Haplotyping indicated that three of the four families with the g10insA mutation were unrelated, indicating that these microinsertions resulted from independent mutational events. Screening of nine f-PCT probands revealed that 44% were heterozygous or homozygous for the common hemochromatosis mutations, which suggests that iron overload may predispose to clinical expression. However, there was no clear correlation between f-PCT disease severity and the URO-D and/or hemochromatosis genotypes. These studies doubled the number of known f-PCT mutations, demonstrated that marked genetic heterogeneity underlies f-PCT, and permitted presymptomatic molecular diagnosis and counseling in these families to enable family members to avoid disease-precipitating factors.

Congenital erythropoietic porphyria: prolonged high-level expression and correction of the heme biosynthetic defect by retroviral-mediated gene transfer into porphyric and erythroid cells.

Congenital erythropoietic porphyria (CEP) is an autosomal recessive disorder resulting from the deficient activity of the heme biosynthetic enzyme uroporphyrinogen III synthase (UROS). Severely affected patients are transfusion dependent and have mutilating cutaneous manifestations. Successful bone marrow transplantation has proven curative, providing the rationale for stem cell gene therapy. Toward this goal, two retroviral MFG vectors containing the UROS cDNA were constructed, one with the wild-type sequence (MFG-UROS-wt) and a second with an optimized Kozak consensus sequence (MFG-UROS-K). Following transduction of CEP fibroblasts, the MFG-UROS-wt and MFG-UROS-K vectors increased the endogenous activity without selection to levels that were 18- and 5-fold greater, respectively, than the mean activity in normal fibroblasts. Notably, the MFG-UROS-wt vector expressed UROS activity in CEP fibroblasts at these high levels for over 6 months without cell toxicity. Addition of either delta-aminolevulinic acid (ALA) or ferric chloride did not affect expression of the transduced UROS gene nor did the increased concentrations of uroporphyrin isomers or porphyrin intermediates affect cell viability. Similarly, transduction of CEP lymphoblasts with the MFG-UROS-wt vector without G418 selection increased the endogenous UROS activity by 7-fold or almost 2-fold greater than that in normal lymphoblasts. Transduction of K562 erythroleukemia cells by cocultivation with the MFG-UROS-wt producer cells increased their high endogenous UROS activity by 1.6-fold without selection. Clonally isolated K562 cells expressed UROS for over 4 months at mean levels 4.7-fold greater than the endogenous activity without cell toxicity. Thus, the prolonged, high-level expression of UROS in transduced CEP fibroblasts and lymphoblasts, as well as in transduced K562 erythroid cells, demonstrated that the enzymatic defect in CEP cells could be corrected by retroviral-mediated gene therapy without selection and that the increased intracellular porphyrin intermediates were not toxic to these cells, even when porphyrin production was stimulated by supplemental ALA or iron. These in vitro studies provide the rationale for ex vivo stem cell gene therapy in severely affected patients with CEP.

Antiproliferative effects of N1,N4-dibenzylputrescine in human and rodent tumor cells.

Polyamines (putrescine, spermidine and spermine) increase in proliferating tissues and are essential for cellular growth and cell division processes. We had previously shown that alkyl substituted putrescines can inhibit cell proliferation. We now tested the effects of the (N(alpha),N(omega)-dibenzyl derivatives of the simple diamines putrescine, cadaverine and 1,3-diaminopropane on the growth of three human squamous cell carcinoma (SCC) lines and a rat hepatoma (H-4-II-E) cell line. Survival assays were measured by treating exponentially-growing SCC cultures with N1,N4-dibenzylputrescine (DBP) (270 microM) or a rat hepatoma cell culture with DBP (100 microM) for 48 hrs. Inhibition of cell growth was measured either by the colony forming assay or by cell counting. DBP inhibited proliferation of the rat hepatoma (H-4-II-E) cell line and induced cytotoxicity when used at a concentration of 100 microM for >48 hrs. N1,N5-dibenzylcadaverine (DBC) also induced cytotoxicity at a similar concentration, while N1,N3-dibenzyl-1,3-diaminopropane (DBPr) was a much weaker inhibitor of cell growth. Inhibition of cell growth by DBP resulted in marked modifications of cell morphology, such as vacuole formation, decrease in size, pycnosis, change in staining behavior toward trypan blue and lack of adherence. DBP was also growth inhibitory in the three human SCC cell lines tested. The concentration of DBP required to achieve growth inhibition of SCC cells could be dramatically decreased in the presence of N1,N4-bis(buta-2,3-dienyl)butanediamine, a specific inhibitor of polyamine oxidase (PAOI). Moreover, although the presence of PAOI only prevented the oxidation (debenzylation) of approximately 20% of intracellular DBP over a 5-day period, it produced a 5-fold increase in the inhibition of cell proliferation by DBP. DBP (and DBC) inhibited putrescine uptake by rat hepatoma (H-4-II-E) cells in what appears to be a competitive reaction. A tenfold excess of putrescine over DBP did not inhibit the antiproliferative or cytotoxic effects of the latter. DBP administered for 72 hrs. depleted intracellular levels of putrescine, spermidine and spermine in the SCC lines by 50-100% of control values. It was found that DBP inhibited nucleic acid and protein synthesis at an early stage of cell proliferation, hence its growth inhibitory effect may be related to inhibition of the synthesis of macromolecules.

Synthesis of isoornithines and methylputrescines. An evaluation of their inhibitory effects on ornithine decarboxylase.

2-(Aminomethyl)-4-aminobutyric acid (isoornithine), 3-methylisoornithine, and 2,3-dimethylisoornithine were not decarboxylated by liver ornithine decarboxylase (ODC, EC 4.1.1.17) of thioacetamide-treated rats but were good competitive inhibitors of the enzyme (Ki ranged from 0.72 to 1.79 mM). When assayed in vivo in the treated rats, the above mentioned isoornithines were also found to inhibit liver ODC when administered 1 h before sacrifice. When the methylputrescines formally derived from the decarboxylation of several isoornithines were assayed on rat liver ODC, it was found that only 2,3-dimethylputrescine decreased the enzymatic activity. When assayed in vivo, it was found to decrease ODC activity by 60%, when the latter was measured 1 h after administration. The effect was reverted 4 h after administration of the drug. Isoornithines were not taken up by H-35 hepatoma cells; hence they did not affect their ODC activity. 2,3-Dimethylputrescine however, was transported into the cells and significantly decreased its ODC activity.