Genetic Analysis of Two Novel GPI Variants Disrupting H Bonds and Localization Characteristics of 55 Gene Variants Associated with Glucose-6-phosphate Isomerase Deficiency.

OBJECTIVE: Glucose-6-phosphate isomerase (GPI) deficiency is a rare hereditary nonspherocytic hemolytic anemia caused by GPI gene variants. This disorder exhibits wide heterogeneity in its clinical manifestations and molecular characteristics, often posing challenges for precise diagnoses using conventional methods. To this end, this study aimed to identify the novel variants responsible for GPI deficiency in a Chinese family. METHODS: The clinical manifestations of the patient were summarized and analyzed for GPI deficiency phenotype diagnosis. Novel compound heterozygous variants of the GPI gene, c.174C>A (p.Asn58Lys) and c.1538G>T (p.Trp513Leu), were identified using whole-exome and Sanger sequencing. The AlphaFold program and Chimera software were used to analyze the effects of compound heterozygous variants on GPI structure. RESULTS: By characterizing 53 GPI missense/nonsense variants from previous literature and two novel missense variants identified in this study, we found that most variants were located in exons 3, 4, 12, and 18, with a few localized in exons 8, 9, and 14. This study identified novel compound heterozygous variants associated with GPI deficiency. These pathogenic variants disrupt hydrogen bonds formed by highly conserved GPI amino acids. CONCLUSION: Early family-based sequencing analyses, especially for patients with congenital anemia, can help increase diagnostic accuracy for GPI deficiency, improve child healthcare, and enable genetic counseling.

Application of Next-Generation Sequencing in Infections After Allogeneic Haematopoietic Stem Cell Transplantation: A Retrospective Study.

This retrospective study aimed to determine the characteristics of infection and diagnostic efficacy of next-generation sequencing (NGS) in patients with fever after allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 71 patients with fever after HSCT were enrolled in this study. Compared with conventional microbiological test (CMT), we found that the sensitivity of NGS versus CMT in peripheral blood samples was 91.2% vs. 41.2%, and that NGS required significantly less time to identify the pathogens in both monomicrobial infections (P=0.0185) and polymicrobial infections (P= 0.0027). The diagnostic performance of NGS was not affected by immunosuppressant use. Viruses are the most common pathogens associated with infections. These results indicated that the sensitivity, timeliness, and clinical significance of NGS are superior for the detection of infections. Although NGS has the advantage of identifying a wide range of potential pathogens, the positive rate is related closely to the sample type. Therefore, we recommend that, in the clinical application of NGS to detect pathogens in patients after allo-HSCT, an appropriate sample type and time should be selected and submitted to improve the positive rate and accuracy of NGS. NGS holds promise as a powerful technology for the diagnosis of fever after HSCT.

Case Report: Is Surgical Treatment Beneficial for Intracranial Basal Ganglia Cunninghamellamycosis Following Haematopoietic Stem Cell Transplantation?

Cunninghamellamycosis is an unusual but often highly fatal mucormycosis caused by Cunninghamella bertholletiae, which belongs to the basal lineage order Mucorales. It is especially fatal when the central nervous system is involved. So far, there are few reported cases of surgical treatment for intracranial mucormycosis in children after allogeneic haematopoietic stem cell transplantation (HSCT). The surgical management of deep-seated basal ganglia fungal lesions remains controversial, and its clinical benefits are not yet well established. Herein, we present a rare case of disseminated mucormycosis caused by C. bertholletiae involving the lung and intracranial basal ganglia after homologous leucocytic antigen-matched sibling donor HSCT. The patient was successfully treated for intracranial cunninghamellamycosis with neuroendoscopic surgery and systemic wide-spectrum antifungal treatment and achieved pulmonary recovery without recurrent C. bertholletiae infection or neurologic sequelae. Over the follow-up period of 13 months, there were no adverse events associated with the intracranial surgical debridement, and the patient remained in good health.

Mechanisms of Senescence-Related NKG2D Ligands Release and Immune Escape Induced by Chemotherapy in Neuroblastoma Cells.

Chemotherapy-induced senescence promotes immunocyte aggregation in the tumor microenvironment by upregulating the surface expression of activating ligands in cancer cells. However, these senescent tumor cells cannot be completely cleared and can induce tumor recurrence. Previous studiesshowed that soluble natural killer (NK) group 2D (NKG2D) ligands impair the recognition of multiple immune cells. In this study, we established an in vitro senescence model using neuroblastoma cells subjected to low-dose Chemotherapeutic drug doxorubicin or the Aurora A inhibitor MLN8237. The results showed that different neuroblastoma cell lines showed increased secretion of the NKG2D ligand MHC class I polypeptide-related sequence A/B (MICA/B) following proteolysis after treatment, with MICA/B subsequently recruited to exosomes to downregulate NKG2D expression in NK cells. Interestingly, disintegrin and metalloproteinase domain-containing 10 (ADAM10) was upregulated in senescent tumor cells, and combined treatment with the ADAM10 inhibitor GI254023X and chemotherapeutic drugs inhibited MICA/B secretion and enhanced recognition and killing by NK cells. Additionally, we found that expression of the long noncoding RNA MALAT1 was significantly increased in senescent neuroblastoma cells, and that MALAT1 served as a sponge for microRNA (miR)-92a-3p to counteract miR-92a-3p-mediated repression of ADAM10 levels. Furthermore, administration of a MALAT1 inhibitor or an miR-92a-3p mimic reduced the MICA/B shedding and enhanced recognition and killing by NK cells. These results confirmed that low-dose chemotherapy induces senescence in neuroblastoma cells, and that senescent tumor cells promote the shedding of the NKG2D ligand MICA/B through the MALAT1/miR-92a/ADAM10 axis, thereby contributing to the formation of a suppressive immune microenvironment and promoting immune escape.

Successful low-dose chemotherapy for refractory Epstein-Barr virus-related post-transplant lymphoproliferative disorder following hematopoietic stem cell transplantation in a child with Wiskott-Aldrich syndrome.

We report the first case of a 12-year-old boy with Wiskott-Aldrich syndrome who developed CD20-weakly expressed and CD30-highly expressed Epstein-Barr virus-related post-transplant lymphoproliferative disorder refractory to rituximab treatment. The patient was effectively and safely treated with personalized low-dose chemotherapy and subsequently remained in complete remission for 1 year.

C/EBPß enhances platinum resistance of ovarian cancer cells by reprogramming H3K79 methylation.

Chemoresistance is a major unmet clinical obstacle in ovarian cancer treatment. Epigenetics plays a pivotal role in regulating the malignant phenotype, and has the potential in developing therapeutically valuable targets that improve the dismal outcome of this disease. Here we show that a series of transcription factors, including C/EBPß, GCM1, and GATA1, could act as potential modulators of histone methylation in tumor cells. Of note, C/EBPß, an independent prognostic factor for patients with ovarian cancer, mediates an important mechanism through which epigenetic enzyme modifies groups of functionally related genes in a context-dependent manner. By recruiting the methyltransferase DOT1L, C/EBPß can maintain an open chromatin state by H3K79 methylation of multiple drug-resistance genes, thereby augmenting the chemoresistance of tumor cells. Therefore, we propose a new path against cancer epigenetics in which identifying and targeting the key regulators of epigenetics such as C/EBPß may provide more precise therapeutic options in ovarian cancer.

Prognostic and therapeutic value of disruptor of telomeric silencing-1-like (DOT1L) expression in patients with ovarian cancer.

BACKGROUND: Epigenetics has been known to play a critical role in regulating the malignant phenotype. This study was designed to examine the expression of DOT1L (histone 3 lysine 79 methyltransferase) and H3K79 methylation in normal ovarian tissues and ovarian tumors and to explore the function of DOT1L and its underline mechanisms in ovarian cancer. METHODS: The expression of DOT1L and H3K79 methylation in 250 ovarian tumor samples and 24 normal ovarian samples was assessed by immunohistochemistry. The effects of DOT1L on cell proliferation in vitro were evaluated using CCK8, colony formation and flow cytometry. The DOT1L-targeted genes were determined using chromatin immune-precipitation coupled with high-throughput sequencing (ChIP-seq) and ChIP-PCR. Gene expression levels were measured by real-time PCR and immunoblotting. The effects of DOT1L on tumor growth in vivo were evaluated using an orthotopic ovarian tumor model. RESULTS: DOT1L expression and H3K79 methylation was significantly increased in malignant ovarian tumors. High DOT1L expression was associated with International Federation of Gynecology and Obstetrics (FIGO) stage, histologic grade, and lymphatic metastasis. DOT1L was an independent prognostic factor for the overall survival (OS) and progression-free survival (PFS) of ovarian cancer, and higher DOT1L expression was associated with poorer OS and PFS. Furthermore, DOT1L regulates the transcription of G1 phase genes CDK6 and CCND3 through H3K79 dimethylation; therefore, blocking DOT1L could result in G1 arrest and thereby impede the cell proliferation in vitro and tumor growth in vivo. CONCLUSIONS: Our findings first demonstrate that DOT1L over-expression has important clinical significance in ovarian cancer and also clarify that it drives cell cycle progression through transcriptional regulation of CDK6 and CCND3 through H3K79 methylation, suggesting that DOT1L might be potential target for prognostic assessment and therapeutic intervention in ovarian cancer.

SIX1 coordinates with TGFß signals to induce epithelial-mesenchymal transition in cervical cancer.

Epithelial-mesenchymal transition (EMT) plays a critical role in promoting tumor invasion and metastasis. However, the key cofactors that modulate the signal transduction to induce EMT have note been fully explored to date. The present study reports that sine oculis homeobox homolog 1 (SIX1) is able to promote EMT of cervical cancer by coordinating with transforming growth factor (TGF)ß-SMAD signals. The expression of SIX1 was negatively correlated with the expression of the epithelial marker E-cadherin in two independent groups of cervical cancer specimens. SIX1 could promote the transition of mesenchymal phenotype in the presence of active TGFß signals in vitro and in vivo. TGFß-SMAD signals were required for the SIX1-mediated promotion of EMT and metastatic capacity of cervical cancer cells. Together, SIX1 and TGFß cooperated to induce more remarkable changes in the transition of phenotype than each of them alone, and coordinated to promote cell motility and tumor metastasis in cervical cancer. These results suggest that the coordination of SIX1 and TGFß signals may be crucial in the EMT program, and that SIX1/TGFß may be considered a valuable marker for evaluating the metastatic potential of cervical cancer cells, or a therapeutic target in the treatment of cervical cancer.

Pharmacodynamics (PD) and pharmacokinetics (PK) of E7389 (eribulin, halichondrin B analog) during a phase I trial in patients with advanced solid tumors: a California Cancer Consortium trial.

BACKGROUND: The California Cancer Consortium completed a phase I trial of E7389 (eribulin mesylate), an analog of the marine natural product halichondrin B. This trial was to determine the pharmacodynamics, pharmacokinetics, and MTD of E7389 administered by bolus injection weekly for 3 weeks out of four. METHODS: This trial included a rapid titration design. Real-time pharmacokinetics were utilized to guide dose escalation. Initially, single-patient cohorts were enrolled with intra- and inter-patient dose doubling. The second phase was a standard 3 + 3 dose escalation schedule. At the MTD, a cohort of patients was enrolled for target validation studies (separate manuscript). The starting dose was 0.125 mg/m(2), and doses were doubled within and between patients in the first phase. Blood and urine sampling for E7389 pharmacokinetics was performed on doses 1 and 3 of cycle 1. Levels were determined using a LC/MS/MS assay. RESULTS: Forty patients were entered. Thirty-eight were evaluable for toxicity and 35 for response. The rapid escalation ended with a grade 3 elevation of alkaline phosphatase at 0.5 mg/m(2)/week. The second phase ended at 2.0 mg/m(2)/week with dose-limiting toxicities of grades 3 and 4 febrile neutropenia. Other toxicities included hypoglycemia, hypophosphatemia, and fatigue. The MTD was 1.4 mg/m(2)/week. Responses included four partial responses (lung cancer [2], urothelial [1], and melanoma [1]). CONCLUSIONS: E7389 was well tolerated in this trial with the major toxicity being myelosuppression. PD shows that E7389 induces significant morphologic changes (bundle formation) in the microtubules of peripheral blood mononuclear cells and tumor cells in vivo. The data suggest that lower intra-tumoral levels of ß-tubulin III or higher intra-tumoral levels of MAP4 may correlate with response to E7389, while lower intra-tumoral levels of stathmin may be associated with progression. PK data reveal that E7389 exhibits a tri-exponential elimination from the plasma of patients receiving a rapid i.v. infusion. At sub-toxic doses, plasma concentrations of E7389 are maintained well above the levels required for activity in vitro for >72 h.

SIX1 promotes tumor lymphangiogenesis by coordinating TGFß signals that increase expression of VEGF-C.

Lymphatic vessels are one of the major routes for the dissemination of cancer cells. Malignant tumors release growth factors such as VEGF-C to induce lymphangiogenesis, thereby promoting lymph node metastasis. Here, we report that sine oculis homeobox homolog 1 (SIX1), expressed in tumor cells, can promote tumor lymphangiogenesis and lymph node metastasis by coordinating with TGFß to increase the expression of VEGF-C. Lymphangiogenesis and lymph node metastasis in cervical cancer were closely correlated with higher expression of SIX1 in tumor cells. By enhancing VEGF-C expression in tumor cells, SIX1 could augment the promoting effect of tumor cells on the migration and tube formation of lymphatic endothelial cells (LEC) in vitro and lymphangiogenesis in vivo. SIX1 enhanced TGFß-induced activation of SMAD2/3 and coordinated with the SMAD pathway to modulate VEGF-C expression. Together, SIX1 and TGFß induced much higher expression of VEGF-C in tumor cells than each of them alone. Despite its effect in promoting VEGF-C expression, TGFß could inhibit lymphangiogenesis by directly inhibiting tube formation by LECs. However, the increased production of VEGF-C not only directly promoted migration and tube formation of LECs but also thwarted the inhibitory effect of TGFß on LECs. That is, tumor cells that expressed high levels of SIX1 could promote lymphangiogenesis and counteract the negative effects of TGFß on lymphangiogenesis by increasing the expression of VEGF-C. These findings provide new insights into tumor lymphangiogenesis and the various roles of TGFß signaling in tumor regulation. Our results also suggest that SIX1/TGFß might be a potential therapeutic target for preventing lymph node metastasis of tumor.

Sine oculis homeobox homolog 1 promotes DNA replication and cell proliferation in cervical cancer.

Malignant proliferation is the fundamental trait of tumor cells. The initiation of DNA replication represents a key process for cell proliferation, and has a marked impact on tumorigenesis and progression. Here we report that Sine oculis homeobox homolog 1 (SIX1) functions as a master regulator in DNA replication of cervical cancer cells. The expression of SIX1 was induced by the E7 oncoprotein of human papillomaviruses in cervical intraepithelial neoplasia and cervical cancer. The increase of SIX1 expression resulted in the upregulation of multiple genes related to the initiation of DNA replication, including the genes coding for the proteins in minichromosome maintenance complex (MCM2, MCM3, MCM6), DNA polymerase α-primase complex (POLA1, PRIM1, PRIM2), clamp loader (RFC3, RFC4, RFC5), DNA polymerase δ complex (POLD3) and DNA polymerase ε complex (POLE2). In line with this, the increase of SIX1 expression enhanced DNA synthesis, accelerated G1 to S phase progression, and promoted the proliferation of cervical cancer cells and the growth of cervical cancer. Consistently, knockdown of SIX1 could hamper DNA synthesis, slow down G1 to S phase progression, and suppress tumor cell proliferation and tumor growth. Importantly, SIX1 could more efficiently promote anchorage-independent cell growth. These results suggest that the increase of SIX1 expression could promote tumorigenesis, progression and invasive growth of cervical cancer by promoting DNA replication, and that targeting SIX1 may have significant therapeutic value in cervical cancer treatment.

Sine oculis homeobox homolog 1 promotes α5ß1-mediated invasive migration and metastasis of cervical cancer cells.

Sine oculis homeobox homolog 1 (SIX1) has been supposed to be correlated with the metastasis and poor prognosis of several malignancies. However, the effect of SIX1 on the metastatic phenotype of tumor cells and the underlying mechanisms were still unclear to date. Here we report that SIX1 can promote α5ß1-mediated metastatic capability of cervical cancer cells. SIX1 promoted the expression of α5ß1 integrin to enhance the adhesion capacity of tumor cells in vitro and tumor cell arrest in circulation in vivo. Moreover, higher expression of SIX1 in tumor cells resulted in the increased production of active MMP-2 and MMP-9, up-regulation of anti-apoptotic genes (BCL-XL and BCL2) and down-regulation of pro-apoptotic genes (BIM and BAX), thus promoting the invasive migration and anoikis-resistance of tumor cells. Importantly, blocking α5ß1 abrogated the regulatory effect of SIX1 on the expression of these genes, and also abolished the promotional effect of SIX1 on invasive capability of tumor cells. Furthermore, knock-down of α5 could abolish the promoting effect of SIX1 on the development of metastatic lesions in both experimental and spontaneous metastasis model. Therefore, by up-regulating α5ß1 expression, SIX1 not only promoted the adhesion capacity, but also augmented ECM-α5ß1-mediated regulation of gene expression to enhance the metastatic potential of cervical cancer cells. These results suggest that SIX1/α5ß1 might be considered as valuable marker for metastatic potential of cervical cancer cells, or a therapeutic target in cervical cancer treatment.

Effects of iodonium-class flavin dehydrogenase inhibitors on growth, reactive oxygen production, cell cycle progression, NADPH oxidase 1 levels, and gene expression in human colon cancer cells and xenografts.

Iodonium-class flavoprotein dehydrogenase inhibitors have been demonstrated to possess antiproliferative potential and to inhibit reactive oxygen production in human tumor cells, although the mechanism(s) that explains the relationship between altered cell growth and the generation of reactive oxygen species (ROS) remains an area of active investigation. Because of the ability of these compounds to inhibit the activity of flavoprotein-containing epithelial NADPH oxidases, we chose to examine the effects of several iodonium-class flavoprotein inhibitors on human colon cancer cell lines that express high, functional levels of a single such oxidase (NADPH oxidase 1, or Nox1). We found that diphenyleneiodonium (DPI), di-2-thienyliodonium (DTI), and iodonium diphenyl inhibited the growth of Caco2, HT-29, and LS-174T colon cancer cells at concentrations (10-250nM for DPI, 0.5-2.5µM for DTI, and 155nM to 10µM for iodonium diphenyl) substantially lower than needed for DU145 human prostate cancer cells, which do not possess functional NADPH oxidase activity. Drug treatment was associated with decreased H2O2 production and diminished intracellular ROS levels, lasting up to 24h, after short-term (1-h) exposure to the iodonium analogs. Decreased tumor cell proliferation was caused, in part, by a profound block in cell cycle progression at the G1/S interface in both LS-174T and HT-29 cells exposed to either DPI or DTI; and the G1 block was produced, for LS-174T cells, by upregulation of p27 and a drug concentration-related decrease in the expression of cyclins D1, A, and E that was partially prevented by exogenous H2O2. Not only did DPI and DTI decrease intracellular ROS, they both also significantly decreased the mRNA expression levels of Nox1, potentially contributing to the prolonged reduction in tumor cell reactive oxygen levels. We also found that DPI and DTI significantly decreased the growth of both HT-29 and LS-174T human tumor xenografts, at dose levels that produced peak plasma concentrations similar to those utilized for our in vitro experiments. These findings suggest that iodonium analogs have therapeutic potential for NADPH oxidase-containing human colon cancers in vivo and that at least part of their antineoplastic mechanism of action may be related to targeting Nox1.

Phase I study of nelfinavir in liposarcoma.

PURPOSE: HIV protease inhibitors are associated with HIV protease inhibitor-related lipodystrophy syndrome. We hypothesized that liposarcomas would be similarly susceptible to the apoptotic effects of an HIV protease inhibitor, nelfinavir. METHODS: We conducted a phase I trial of nelfinavir for liposarcomas. There was no limit to prior chemotherapy. The starting dose was 1,250 mg twice daily (Level 1). Doses were escalated in cohorts of three to a maximally evaluated dose of 4,250 mg (Level 5). One cycle was 28 days. Steady-state pharmacokinetics (PKs) for nelfinavir and its primary active metabolite, M8, were determined at Levels 4 (3,000 mg) and 5. RESULTS: Twenty subjects (13 males) were enrolled. Median (range) age was 64 years (37-81). One subject at Level 1 experienced reversible, grade 3 pancreatitis after 1 week and was replaced. No other dose-limiting toxicities were observed. Median (range) number of cycles was 3 (0.6-13.5). Overall best responses observed were 1 partial response, 1 minor response, 4 stable disease, and 13 progressive disease. Mean peak plasma levels and AUCs for nelfinavir were higher at Level 4 (7.3 mg/L; 60.9 mg/L × h) than 5 (6.3 mg/L; 37.7 mg/L × h). The mean ratio of M8:nelfinavir AUCs for both levels was ~1:3. CONCLUSIONS: PKs demonstrate auto-induction of nelfinavir clearance at the doses studied, although the mechanism remains unclear. Peak plasma concentrations were within range where anticancer activity was demonstrated in vitro. M8 metabolite is present at ~1/3 the level of nelfinavir and may also contribute to the anticancer activity observed.

Single-dose pharmacokinetic and toxicity analysis of pyrrole-imidazole polyamides in mice.

PURPOSE: Pyrrole-imidazole (Py-Im) polyamides are programmable, sequence-specific DNA minor groove-binding ligands. Previous work in cell culture has shown that various polyamides can be used to modulate the transcriptional programs of oncogenic transcription factors. In this study, two hairpin polyamides with demonstrated activity against androgen receptor signaling in cell culture were administered to mice to characterize their pharmacokinetic properties. METHODS: Py-Im polyamides were administered intravenously by tail vein injection. Plasma, urine, and fecal samples were collected over a 24-h period. Liver, kidney, and lung samples were collected postmortem. Concentrations of the administered polyamide in the plasma, excretion, and tissue samples were measured using LC/MS/MS. The biodistribution data were analyzed by both non-compartmental and compartmental pharmacokinetic models. Animal toxicity experiments were also performed by monitoring weight loss after a single subcutaneous (SC) injection of either polyamide. RESULTS: The biodistribution profiles of both compounds exhibited rapid localization to the liver, kidneys, and lungs upon injection. Plasma distribution of the two compounds showed distinct differences in the rate of clearance, the volume of distribution, and the AUCs. These two compounds also have markedly different toxicities after SC injection in mice. CONCLUSIONS: The variations in pharmacokinetics and toxicity in vivo stem from a minor chemical modification that is also correlated with differing potency in cell culture. The results obtained in this study could provide a structural basis for further improvement of polyamide activity both in cell culture and in animal models.

Nelfinavir induces liposarcoma apoptosis through inhibition of regulated intramembrane proteolysis of SREBP-1 and ATF6.

PURPOSE: We previously reported that nelfinavir (NFV) induces G(1) cell-cycle block and apoptosis selectively in liposarcoma cell lines due to increased SREBP-1 (sterol regulatory element binding protein-1) expression in the absence of increased transcription. We postulate that NFV interferes with regulated intramembrane proteolysis of SREBP-1 and ATF6 (activating transcription factor 6). EXPERIMENTAL DESIGN: Time-lapse, confocal microscopic studies show that NFV inhibits the nuclear translocation of full-length SREBP-1-EGFP and ATF6-EGFP fusion proteins. siRNA-mediated knockdown of site-1 protease (S1P) and/or site-2 protease (S2P) leads to inhibition of SREBP-1 intracellular trafficking to the nucleus and reduces liposarcoma cell proliferation. Treatment of LiSa-2 liposarcoma cells with 3,4-dichloroisocoumarin, a serine protease inhibitor of S1P, did not affect SREBP-1 processing. In contrast, 1,10-phenanthroline, an S2P-specific inhibitor, reproduces the molecular and biological phenotypes observed in NFV-treated cells, which implicates S2P as a target of NFV. In vivo evaluation of NFV in a murine liposarcoma xenograft model leads to inhibition of tumor growth without significant toxicity. RESULTS: NFV-induced upregulation of SREBP-1 and ATF6 results from inhibition of S2P, which together with S1P mediates regulated intramembrane proteolysis from their precursor to their transcriptionally active forms. The resulting endoplasmic reticulum (ER) stress and concurrent inhibition of the unfolded protein response induce caspase-mediated apoptosis. CONCLUSIONS: These results provide new insight into the mechanism of NFV-mediated induction of ER stress and cell death in liposarcomas and are the first to report targeting S2P for cancer therapy.

Structural basis on the dityrosyl-diiron radical cluster and the functional differences of human ribonucleotide reductase small subunits hp53R2 and hRRM2.

Ribonucleotide reductase (RNR) is an enzyme for the de novo conversion of ribonucleotides to deoxyribonucleotides. The two human RNR small subunits hRRM2 and hp53R2 share 83% sequence homology but show distinct expression patterns and function. Structural analyses of the oxidized form of hRRM2 and hp53R2 indicate that both proteins contain a conserved Gln127-hp53R2/Gln165-hRRM2 close to the dinuclear iron center and the essential tyrosine residue Tyr124-hp53R2/Tyr162-hRRM2 forms hydrogen bonds with the tyrosine and iron ligands, implying a critical role for the glutamine residue in assembling the dityrosyl-diiron radical cofactor. The present work also showed that Tyr221 in hRRM2, which is replaced by Phe183 in hp53R2, forms a hydrogen bond with Tyr162 to extend the hydrogen bond network from Gln165-hRRM2. Mutagenesis and spectroscopic experiments suggested that the tyrosine-to-phenylalanine switch at Phe183-hp53R2/Tyr221-hRRM2 could lead to differences in radical generation or enzymatic activity for hp53R2 and hRRM2. This study correlates the distinct catalytic mechanisms of the small subunits hp53R2 and hRRM2 with a hydrogen-bonding network and provides novel directions for designing and developing subunit-specific therapeutic agents for human RNR enzymes.

Advanced glycation end products of DNA: quantification of N2-(1-Carboxyethyl)-2'-deoxyguanosine in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry.

Methylglyoxal (MG) and related alpha-oxoaldehydes react with proteins, lipids, and DNA to give rise to covalent adducts known as advanced glycation end products (AGEs). Elevated levels of AGEs have been implicated in the pathological complications of diabetes, uremia, Alzheimer's disease, and possibly cancer. There is therefore widespread interest in developing sensitive methods for the in vivo measurement of AGEs as prognostic biomarkers and for treatment monitoring. The two diastereomeric MG-DNA adducts of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (CEdG) are the primary glycation products formed in DNA; however, accurate assessment of their distribution in vivo has not been possible since there is no readily available quantitative method for CEdG determination in biological samples. To address these issues, we have developed a sensitive and quantitative liquid chromatography electrospray ionization tandem mass spectrometry assay using the stable isotope dilution method with an (15)N(5)-CEdG standard. Methods for CEdG determination in urine or tissue extracted DNA are described. Changes in urinary CEdG in diabetic rats in response to oral administration of the AGE inhibitor LR-90 are used to demonstrate the potential utility of the method for treatment monitoring. Both stereoisomeric CEdG adducts were detected in a human breast tumor and normal adjacent tissue at levels of 3-12 adducts/10(7) dG, suggesting that this lesion may be widely distributed in vivo. Strategies for dealing with artifactual adduct formation due to oxoaldehyde generation during DNA isolation and enzymatic workup procedures are described.

Oblimersen and alpha-interferon in metastatic renal cancer: a phase II study of the California Cancer Consortium.

PURPOSE: Oblimersen is an 18-base oligodeoxynucleotide encoding antisense to the gene for bcl-2, an anti-apoptotic protein that is upregulated in renal and other cancers. This study was designed to evaluate the combination of oblimersen with alpha-Interferon in advanced renal cancer. Trial endpoints were antitumor efficacy and toxicity, pharmacokinetics, and evidence of apoptosis in peripheral blood mononuclear cells. METHODS: Patients with measurable advanced renal cancer and 0-1 prior therapy were eligible. Treatment consisted of oblimersen, 7 mg/kg/day, as a continuous intravenous infusion 7 days of every 14 day cycle, plus alpha-IFN, 5 million units/m(2) subcutaneously, days 4 and 6 of the first oblimersen infusion, then thrice weekly. Blood for laboratory correlates was collected before treatment, during oblimersen, and during therapy with both agents. RESULTS: Twenty-three patients were enrolled, five of whom had prior systemic therapy (three with prior high-dose interleukin-2). The median number of treatment cycles was 4 (range 1-12). One patient had a partial response lasting 2.5 months. The Grade 3-4 toxicities were fatigue, fever, myelosuppression, hepatic enzyme and metabolic abnormalities. Laboratory analyses of CD3+ lymphocyte apoptotic markers demonstrated no change between pre-treatment and on-treatment levels of bcl-2 or Annexin/PI positivity by flow cytometry. Mean oblimersen steady-state plasma concentration and clearance was 2.3 +/- 0.9 microg/ml and 0.15 +/- 0.07 l/h/kg, respectively. CONCLUSIONS: Oblimersen given in this dose and schedule with alpha-IFN does not appear sufficiently active to warrant further study in advanced renal cancer. Combinations with newer targeted agents may show greater promise.