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Purpose: VEGF and infiltrating myeloid cells are known regulators of tumor angiogenesis and vascular permeability in glioblastoma. We investigated potential blood-based markers associated with radiographic changes to aflibercept, which binds VEGF and placental growth factor (PlGF) in patients with recurrent glioblastoma.Experimental Design: In this single-arm phase II trial, aflibercept was given intravenously every two weeks until disease progression. Plasma and peripheral blood mononuclear cells were collected at baseline and 24 hours, 14 days, and 28 days posttreatment. Plasma cytokines and angiogenic factors were quantified by using ELISA and multiplex bead assays, and myeloid cells were assessed by flow cytometry in a subset of patients.Results: Circulating levels of VEGF significantly decreased 24 hours after treatment with aflibercept, coincident with radiographic response observed by MRI. PlGF initially decreased 24 hours posttreatment but increased significantly by days 14 and 28. Lower baseline levels of PlGF, elevated baseline levels of CTACK/CCL27, MCP3/CCL7, MIF, and IP-10/CXCL10, and a decrease in VEGFR1+ monocytes from baseline to 24 hours were all associated with improved response. Tumor progression was associated with increases in circulating matrix metalloproteinase 9.Conclusions: These data suggest that decreases in VEGF posttreatment are associated with radiographic response to aflibercept. Elevated baseline chemokines of monocyte lineage in responding patients supports a role for myeloid cells and chemokines as potential biomarkers and regulators of glioma angiogenesis. Clin Cancer Res; 17(14); 4872–81. ©2011 AACR.

Supplementary Figure 1 from Myeloid Biomarkers Associated with Glioblastoma Response to Anti-VEGF Therapy with Aflibercept

PDF file - 86KB, Quantitative analysis of CTEC and CEC/CEP populations in human specimens by eight-color flow cytometry. A, Syto16 (DNA) gate used to exclude apoptotic, necrotic debris and cellular degraded fragments. Syto16-positive cells (red and blue dots) indicate all viable portions in peripheral blood mononuclear cells (PBMCs) specimen. B, P1 gate with SSC/FSC to display major blood components of the Syto16+ PBMCs (green dots). C, CD45-negative cells gated on Syto16/P1 to specifically select non-hematopoietic cells (blue dots) for further phenotypic analysis and quantitative determination. D. CD133 rectangle gate made on Syto16/P1/CD45-negative population to separate CD45-/CD133+ (pink dots) and CD45-/CD133- (blue dots) subsets. E and F, CECs/CEPs gated on Syto16/P1/CD45-/CD133+ or Syto16/P1/CD45-/CD133- subsets respectively, illustrate staining negatively or dim for hematopoietic marker CD45, positively for SYTO16 (DNA), endothelial markers CD146 and CD31, and negatively for the progenitor marker CD133 (pan dots). CEPs expressed the same phenotype as CECs except they were positively staining for CD133 (pink dots). G, Quadrant gates made on Syto16/P1/CD45- to identify the subsets expressing CD276+/CD133- (blue dots) for further phenotypic analysis and CTECs quantitative determination. H, CTECs gated on Syto16/P1/CD45-/CD133-/CD276+ demonstrate staining negatively or dim for hematopoietic marker CD45, positively for SYTO16 (DNA), tumor-derived endothelial marker CD276, endothelial markers CD146 and CD31, and negatively for the progenitor marker CD133 (aqua dots).

PDF file - 104KB, Quantitative analysis of CTEC and CEC/CEP populations in mouse specimens by flow cytometry. Syto16 (DNA) gate used to exclude apoptotic, necrotic debris and cellular degraded fragments. CECs were characterized as CD31+CD45-VEGFR-2+CD117-, and CEPs as CD31+CD45-VEGFR-2+CD117+. Gating of CD276+ (top) and TEM7+ (bottom) CTECs from a representative animal specimen is shown.

PDF file - 40KB, CECs and CEPs in peripheral blood of normal and tumor-bearing mice were evaluated using flow cytometry. CTECs were first identified based on the staining pattern of CECs and further characterized using CD276 and TEM7 antibodies. (A) Percentage of CECs positive for CD276. (B) Percentage of CECs positive for TEM7. * p

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Background: Sunitinib is approved worldwide for treatment of advanced pancreatic neuroendocrine tumours (pNET), but no validated markers exist to predict response. This analysis explored biomarkers associated with sunitinib activity and clinical benefit in patients with pNET and carcinoid tumours in a phase II study. Methods: Plasma was assessed for vascular endothelial growth factor (VEGF)-A, soluble VEGF receptor (sVEGFR)-2, sVEGFR-3, interleukin (IL)-8 (n=105), and stromal cell-derived factor (SDF)-1α (n=28). Pre-treatment levels were compared between tumour types and correlated with response, progression-free (PFS), and overall survival (OS). Changes in circulating myelomonocytic and endothelial cells were also analysed. Results: Stromal cell-derived factor-1α and sVEGFR-2 levels were higher in pNET than in carcinoid (P=0.003 and 0.041, respectively). High (above-median) baseline SDF-1α was associated with worse PFS, OS, and response in pNET, and high sVEGFR-2 with longer OS (P⩽0.05). For carcinoid, high IL-8, sVEGFR-3, and SDF-1α were associated with shorter PFS and OS, and high IL-8 and SDF-1α with worse response (P⩽0.05). Among circulating cell types, monocytes showed the largest on-treatment decrease, particularly CD14+ monocytes co-expressing VEGFR-1 or CXCR4. Conclusions: Interleukin-8, sVEGFR-3, and SDF-1α were identified as predictors of sunitinib clinical outcome. Putative pro-tumorigenic CXCR4+ and VEGFR-1+ monocytes represent novel candidate markers and biologically relevant targets explaining the activity of sunitinib.

Background Preoperative anaemia is associated with increased morbidity in patients undergoing major surgery. Whether erythrocytes are the only bone-marrow-derived cell lineage that associates with increased surgical complications is unknown. This prospective observational trial studied the mobilization of endothelial progenitor cells (EPCs) in response to exercise in association with postoperative complications. Methods After IRB approval, 60 subjects undergoing major thoracic surgery were exercised to exhaustion (peak V ˙ O 2 ). Peripheral blood collected before and after peak exercise was quantified for EPC lineages by fluorescence-activated cell sorter analysis. Complication analysis was based on the Clavien–Dindo classification. Results Exhaustive exercise increased EPC [CD45−133+34+ cells=150 (0.00–5230) to 220 (0.00–1270) cells μl−1; median change (range)=20 (−4,180–860) cells μl−1; P=0.03] but not mature endothelial cell (EC) subpopulations. Pre-exercise levels [odds ratio (OR)=0.86, 95% confidence interval (CI): 0.37–2.00, P=0.72), change after exercise as a continuous variable (OR=0.95, 95% CI: 0.41–2.22, P=0.91) and a positive response after exercise (change >0 cells μl−1; OR=0.41, 95% CI: 0.13–1.28, P=0.12) were not statistically significantly associated with the incidence of postoperative complications. Post-hoc receiver operating characteristic curve analyses revealed that subjects with a CD45−133+34+ increase ≥60 cells μl−1 in response to exercise suffered fewer postoperative complications [86% sensitivity, 48% specificity and AUC=0.67 (95% CI: 0.52–0.81)]. Conclusions Preoperative exercise induces EPC into the peripheral circulation. Subjects with a poor EPC response had a pre-existing propensity for postoperative complications. This warrants further research into the role of bone marrow function as a critical component to endothelial repair mechanisms. Clinical trial registration IRB 2003-0434 (University of Texas M.D. Anderson Cancer Center, Houston, TX, USA).

Circulating endothelial cells (CEC) are derived from multiple sources, including bone marrow (circulating endothelial progenitors; CEP), and established vasculature (mature CEC). Although CECs have shown promise as a biomarker for patients with cancer, their utility has been limited, in part, by the lack of specificity for tumor vasculature and the different nonmalignant causes that can impact CEC. Tumor endothelial markers (TEM) are antigens enriched in tumor versus nonmalignant endothelia. We hypothesized that TEMs may be detectable on CEC and that these circulating TEM+ endothelial cells (CTEC) may be a more specific marker for cancer and tumor response than standard CEC. We found that tumor-bearing mice had a relative increase in numbers of circulating CTEC, specifically with increased levels of TEM7 and TEM8 expression. Following treatment with various vascular-targeting agents, we observed a decrease in CTEC that correlated with the reductions in tumor growth. We extended these findings to human clinical samples and observed that CTECs were present in patients with esophageal cancer and non–small cell lung cancer (N = 40), and their levels decreased after surgical resection. These results demonstrate that CTECs are detectable in preclinical cancer models and patients with cancer. Furthermore, they suggest that CTECs offer a novel cancer-associated marker that may be useful as a blood-based surrogate for assessing the presence of tumor vasculature and antiangiogenic drug activity. Cancer Res; 74(10); 2731–41. ©2014 AACR.

BACKGROUND: Circulating endothelial cells (CECs) are a candidate biomarker for monitoring angiogenesis in cancer. Circulating endothelial cell subsets are mobilised by angiogenic mediators. Because of the highly angiogenic phenotype of renal cell carcinoma (RCC), we sought to assess the potential of CECs as a marker of RCC in patients with von Hippel-Lindau (VHL) disease and those with sporadic RCC. METHODS: We performed multicolour flow cytometry to enumerate CECs in patients with RCC, patients with VHL disease with and without RCC, and normal subjects. Two subsets of CECs were evaluated: mature CECs (mCECs) and circulating endothelial progenitors (CEPs). RESULTS: In patients with VHL disease and RCC and those with sporadic RCC (N ¼10), CEPs and the CEP:mCEC ratio were higher than in normal subjects (N ¼17) (median CEPs: 0.97 vs 0.19 cellsml � 1 , respectively, Po0.01; median CEP:mCEC: 0.92 vs 0.58, respectively, P ¼0.04). However, in patients with VHL without RCC, CECs were not increased. In paired pre- and post-nephrectomy RCC patient samples (N ¼20), CEPs decreased after surgery (median difference 0.02 cellsml � 1 , � 0.06 to 1.2; P ¼0.05). CONCLUSION: Circulating endothelial progenitors were elevated in RCC, but not in patients with VHL without RCC. Circulating endothelial progenitor enumeration merits further investigation as a monitoring strategy for patients with VHL.

Purpose: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) gefitinib and erlotinib benefit some non–small cell lung cancer (NSCLC) patients, but most do not respond (primary resistance) and those who initially respond eventually progress (acquired resistance). EGFR TKI resistance is not completely understood and has been associated with certain EGFR and K-RAS mutations and MET amplification.Experimental Design: We hypothesized that dual inhibition of the vascular endothelial growth factor (VEGF) and EGFR pathways may overcome primary and acquired resistance. We investigated the VEGF receptor/EGFR TKI vandetanib, and the combination of bevacizumab and erlotinib in vivo using xenograft models of EGFR TKI sensitivity, primary resistance, and three models of acquired resistance, including models with mutated K-RAS and secondary EGFR T790M mutation.Results: Vandetanib, gefitinib, and erlotinib had similar profiles of in vitro activity and caused sustained tumor regressions in vivo in the sensitive HCC827 model. In all four resistant models, vandetanib and bevacizumab/erlotinib were significantly more effective than erlotinib or gefitinib alone. Erlotinib resistance was associated with a rise in both host and tumor-derived VEGF but not EGFR secondary mutations in the KRAS mutant-bearing A549 xenografts. Dual inhibition reduced tumor endothelial proliferation compared with VEGF or EGFR blockade alone, suggesting that the enhanced activity of dual inhibition is due at least in part to antiendothelial effects.Conclusion: These studies suggest that erlotinib resistance may be associated with a rise in both tumor cell and host stromal VEGF and that combined blockade of the VEGFR and EGFR pathways can abrogate primary or acquired resistance to EGFR TKIs. This approach merits further evaluation in NSCLC patients.

In recent years, there has been significant progress in the clinical development and application of antiangiogenic therapies in renal cell carcinomas, particularly inhibitors of the vascular endothelial growth factor (VEGF) pathway. Despite this progress, no validated methods are currently available for identifying which patients are most likely to respond to treatment or experience toxic effects, selecting the optimal dose, or determining whether the intended molecular target has been effectively inhibited. However, recent studies have suggested that some of the biomarkers currently under investigation in clear cell renal cell carcinoma for VEGF pathway inhibitors are promising. These biomarkers include circulating proangiogenic factors and receptors; markers of hypoxia and endothelial damage; and cellular populations in peripheral blood, such as circulating endothelial cells. Further preclinical and translational validation studies are still needed to determine their practical utility in the clinical setting.

Purpose: Antigenic overlap among circulating endothelial cells (CEC) and progenitors (CEP), platelets, and other blood cells led to the need to develop a reliable standardized method for CEC and CEP quantification. These cells are emerging as promising preclinical/clinical tools to define optimal biological doses of antiangiogenic therapies and to help stratify patients in clinical trials. Experimental Design: We report the experimental validation of a novel flow cytometry method that precisely dissects CEC/CEP from platelets and other cell populations and provides information about CEC/CEP viability. Results: Sorted DNA/Syto16+CD45−CD31+CD146+ CECs, investigated by electron microscopy, were found to be bona fide endothelial cells by the presence of Weibel-Palade bodies. More than 75% of the circulating mRNAs of the endothelial-specific gene, VE-cadherin, found in the blood were present in the sorted population. CECs were 140 ± 171/mL in healthy subjects (n = 37) and 951 ± 1,876/mL in cancer patients (n = 78; P < 0.0001). The fraction of apoptotic/necrotic CECs was 77 ± 14% in healthy subjects and 43 ± 23% in cancer patients (P < 0.0001). CEPs were 181 ± 167/mL in healthy donors and 429 ± 507/mL in patients (P = 0.00019). Coefficients of variation were 4 ± 4% (intrareader), 17 ± 4% (interreader), and 17 ± 7% (variability over 0-72 h), respectively. Parallel samples were frozen by a standardized protocol. After thawing, coefficients of variation were 12 ± 8% (intrareader), 16 ± 10% (interreader), and 26 ± 16% (variability over 0-14 days of frozen storage), respectively. Conclusions: This procedure enumerates a truly endothelial cell population with limited intrareader and interreader variability. It appears possible to freeze samples for large-scale CEC enumeration during clinical trials. This approach could be enlarged to investigate other angiogenic cell populations as well.

Serine Protease Inhibitor Kazal-type 5 (SPINK5) gene encodes 3 different Lympho- Epithelial Kazal-Type-Inhibitor (LEKTI) isoforms which are organized into longer than 15, 15, and 13 inhibitory domains. We identified LEKTI by its constitutive expression in normal oral mucosa and lost or down regulated expression in matched tumor specimens of patients with head and neck squamous cell carcinoma (HNSCC). Previously, we showed that recombinant full-length LEKTI and rLEKTI fragments inhibit the activity of plasmin, subtilisin A, cathepsin G, neutrophil elastase, trypsin, caspase 14, and kallikreins (KLK) 5, 6, 7, 13, and 14 to varied extents. Here, we show that LEKTI protein is absent in HNSCC OSC19, Tu138, Tu177, and UMSCC1 lines. We then determined the consequences of LEKTI re-expression on migration and invasion, adhesion and gene expression profile of HNSCCOSC19 and UMSCC1 lines. We demonstrate that LEKTI expressing OSC19 and UMSCC1 clones show markedly reduced migration and invasion. Moreover, LEKTI expressing OSC19 clones show striking morphological changes and enhanced adhesionon type I, III, IV, and V collagens, fibronectin, and laminin5.In addition, we show that exogenous r-LEKTI blocks migration of OSC19-parental cells in a dose and time dependent manner. Microarray analysis identified 186 genes which are differentially regulated in both OSC19 LEKTI clones. MMP-14, KLK5, and ADAM8 are down regulated while MMP-3, LEKTI, DSC2 and DSC3are up-regulated in OSC19 LEKTI clones. RT-PCR and Western blot results confirmed microarray results for MMP-14 and MMP-3in OSC19 LEKTI clones. In addition we discover that MMP-9 protein expression and pro-MMP-9 activity are severely reduced in LEKTI expressing clones as shown by WB and zymogram. Together, this work provides mechanistic insights into how loss of LEKTI protein expression promotes an invasive phenotype in HNSCC tumors.

The insulin-like growth factor II (IGF2) is a polypeptide with structure homology to insulin which possesses mitogen activity, and imprinted with paternal allele. In order to elucidate the distribution of imprinting pattern and relationship between allele- and tissue-specific expression of IGF2 in growth and maturation of human tissues, we investigated allele-specific expression of IGF2 in a wide spectrum of normal maturated human tissues by a PCR-based assay and found monoallelic expression in all eight-type tissues tested except human adult liver. Moreover, when a RT-PCR based sensitive allele-specific primer extension for an Apa I polymorphism within exon 9 of IGF2 was used, the analysis revealed the gene was normally imprinted in placenta; in contrast to the finding with placenta, IGF2 transcripts were biallelically expressed in human adult liver. Our results have clearly demonstrated preferential paternal expression and tissue-specific imprinting pattern of IGF2 in all human tissues tested in this study. Collectively, since IGF2 expression in developing fetal and adult liver is specified by distinct promoters, these extensive observations definitively indicate that transcriptional imprinting of IGF2 is more likely a promoter dependent manner.

Genomic imprinting is a non-Mendelian form of gene regulation resulting in differential allelic expression of a gene and plays an important role in the development of certain types of cancer and genetic diseases. Imprinting of IGF2 was demonstrated in normal placenta and fetal kidney by showing monoallelic paternal expression. In contrast, several kinds of tumor showed biallelic expression of IGF2, suggesting that relaxation of the normal imprinting of this growth cytokine may be a feature of some tumors. In this study we wished to determine whether relaxation of IGF2 imprinting was also common in human leukemias. An intragenic Apa I polymorphism within the 3' untranslated region of the gene was used to examine allele-specific transcription in leukemic blasts derived from 24 primary patients with acute myeloid leukemia by RT-PCR. Leukemic samples from 12 of the 24 leukemia patients were identified as heterozygous for IGF2 and potentially informative for the RT-PCR assay. RNA-PCR from these informative leukemia samples studied showed biallelic expression of IGF2, indicating loss of normal imprinting of this gene to be very frequent. In conclusion, constitutional loss of monoallelic expression in 50% of the leukemia samples tested suggests that abnormal imprinting of IGF2 could play an important role in either leukemogenesis or cytokine dysregulation for leukemic cells.

hLH-2, a transcription factor that contains double cysteine rich regions (LIM motifs) and a homeobox (Hox) DNA-binding domain shows aberrant high expression in all cases of chronic myelogenous leukemia (CML). This gene has been mapped to the chromosome 9q33–34.1, the same region as the reciprocal translocation that creates the breakpoint cluster region (BCR)-ABL chimera of the Philadelphia chromosome (Ph′). To investigate the possible involvement between the BCR-ABL fusion gene and hLH-2 in the pathogenesis of CML, an hLH-2-negative CML cell line, JK-1 that carries double Ph′ chromosomes, was examined. Like other CML cells, high BCR-ABL fusion mRNA levels are expressed, but no transcript of hLH-2 was detected in JK-1 cells as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Compared with the CML cell line, K-562, an additional rearrangement of the BCR gene was observed in JK-1 as determined by Southern blot hybridization; however, the hLH-2 gene was normal. These findings raise interesting questions about the possible roles of either the abnormal BCR gene or other genetic events such as the complex chromosomal abnormalities that result in hLH-2 being turned off in JK-1 cells.

Flow cytometry is a powerful tool for analyzing multiple parameters of individual cells and facilitating the identification of different cell types within heterogeneous populations. This technique has already become a routine method for diagnosis, prognosis, and monitoring of patients with many types of disorders and a crucial tool for accurately analyzing frequencies and relative values of cellular antigen expression. However, there are many technical obstacles involved in flow cytometric testing. This chapter provides an optimized immunostaining procedure with procedural variation for specific situations and a troubleshooting checklist.

Purpose: VEGF and infiltrating myeloid cells are known regulators of tumor angiogenesis and vascular permeability in glioblastoma. We investigated potential blood-based markers associated with radiographic changes to aflibercept, which binds VEGF and placental growth factor (PlGF) in patients with recurrent glioblastoma. Experimental Design: In this single-arm phase II trial, aflibercept was given intravenously every two weeks until disease progression. Plasma and peripheral blood mononuclear cells were collected at baseline and 24 hours, 14 days, and 28 days posttreatment. Plasma cytokines and angiogenic factors were quantified by using ELISA and multiplex bead assays, and myeloid cells were assessed by flow cytometry in a subset of patients. Results: Circulating levels of VEGF significantly decreased 24 hours after treatment with aflibercept, coincident with radiographic response observed by MRI. PlGF initially decreased 24 hours posttreatment but increased significantly by days 14 and 28. Lower baseline levels of PlGF, elevated baseline levels of CTACK/CCL27, MCP3/CCL7, MIF, and IP-10/CXCL10, and a decrease in VEGFR1+ monocytes from baseline to 24 hours were all associated with improved response. Tumor progression was associated with increases in circulating matrix metalloproteinase 9. Conclusions: These data suggest that decreases in VEGF posttreatment are associated with radiographic response to aflibercept. Elevated baseline chemokines of monocyte lineage in responding patients supports a role for myeloid cells and chemokines as potential biomarkers and regulators of glioma angiogenesis. Clin Cancer Res; 17(14); 4872–81. ©2011 AACR.

Novel treatment approaches are needed for children with advanced neuroblastoma. Studies with neuroblastoma cells have indicated the presence of a hypoxia-driven vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-1 autocrine loop modulating hypoxia-inducible factor-1alpha (HIF-1alpha). Whether other receptor tyrosine kinases (RTKs) are capable of modulating HIF-1alpha levels and whether RTKs can regulate HIF-2alpha as well is largely unknown. We evaluated neuroblastoma cell lines for expression of various RTKs. Although cell lines were heterogeneous in the expression of VEGFR-1, -3, c-Kit and RET, most cells expressed PDGFR-alpha and -beta. Ligand-induced activation of multiple RTKs upregulated HIF-1alpha levels, whereas activation of VEGFR-1 alone upregulated HIF-2alpha. Multitargeted tyrosine kinase inhibitor sunitinib reduced hypoxia-induced rises in HIF-1alpha and HIF-2alpha through mechanisms involving effects on both mRNA levels and protein stability. In addition, sunitinib and sorafenib had direct effects on tumor cell viability in vitro. In a neuroblastoma xenograft model, tumor growth inhibition by sunitinib was associated with inhibition of angiogenesis and reduced HIF-1alpha levels. These findings show that multiple RTKs may regulate the HIF axis in normoxia and hypoxia and suggest that multikinase inhibitors may exert antiangiogenic effects not only by direct effects on endothelial cells, but also by blocking compensatory hypoxia- and ligand-induced changes in HIF-1alpha and HIF-2alpha.

Tumor angiogenesis has been associated with a poor prognosis in patients with metastatic melanoma (MM). Microtubule stabilizers and cyclooxygenase 2 (COX-2) inhibitors, alone and in combination, have produced inhibitory effects on endothelial cells and tumor angiogenesis. Angiogenesis, which is the growth of new blood vessels, is necessary for tumor growth and progression. Thus, the authors tested the safety and efficacy of a low dose of paclitaxel and celecoxib in patients with MM.: Patients received paclitaxel 10 mg/m(2) for 96 hours weekly as a continuous intravenous infusion and oral celecoxib 400 mg twice daily. Systemic tumor response was assessed at 6-week intervals. Tumor measurements at the end of Cycle 1 were used as the baseline for assessment of tumor progression. Patients with unacceptable toxicity or disease progression after Cycle 2 relative to the end of Cycle 1 were taken off study.: Twenty patients were enrolled. Twelve of 20 patients (60%) had receivedor =2 previous systemic therapies. Three patients did not receive treatment because of rapid disease progression. Treatment-related grade 3/4 toxicities were limited to catheter-related complications. One patient achieved a partial response, and 3 of 20 patients (15%) had stable disease for6 months. The median time to progression was 57 days (95% confidence interval, 43-151 days), and the median overall survival was 212 days (95% confidence interval, 147-811 days).: Low-dose, continuous intravenous infusion paclitaxel and oral celecoxib produced disease stabilization in a significant proportion of heavily pretreated patients with MM. These findings support a role for metronomic therapy in patients with this disease. Cancer 2010. (c) 2010 American Cancer Society.

Background Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro . Methods Expression of 12 of the KLKs ( KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities. Results We here describe for the first time expression of numerous KLKs ( KLKs 5–8, 10–14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4–6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro . Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro . Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation. Conclusion We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.

Using clonogenic assay we investigated the effect of stem cell factor (SCF) on the in vitro growth of clonogenic precursor cells from acute myeloid leukemia (AML) and myelodys-plastic syndromes (MDS) in the presence or absence of recombinant human erythropoietin (rhEpo) or recombinant human granulocyte colony-stimulating factor (rhG-CSF). SCF as a single factor did not induce significant colony formation, and even in the presence of rhEpo or rhG-CSF it very weakly stimulated erythroid colony formation and was rarely capable of inducing myeloid colony formation by clonogenic leukemic cells. In culture dishes supplemented with SCF, both myeloid and erythroid colony formations were dramatically enhanced in MDS, regarding both colony number and size. Cotony-formation abilities by MDS progenitors were improved following costimulation with SCF and rhEpo. These results suggest that SCF may have a therapeutic role in restoring hematopoiesis in patients with MDS. © 1994 Wiley-Liss, Inc.

Breast cancer is the most frequent malignancy in women and genetically heterogeneous, and a variety of genetic lesions have been identified that tend to accumulate during the disease progress. In breast cancer, loss of heterozygosity (LOH) has been described in the critical regions of chromosomes 11p15 and 11q22-23. Genomic imprinting is defined as gamete specific modification causing differential expression of the two alleles of a gene, in somatic cells. Human insulin like growth factor II gene (IGF2), located on chromosome 11p15, the same region on which LOH frequently occurred in breast cancer, has been recently identified as a genomic imprinting gene expressing preferentially paternal allele. To determine whether loss of IGF2 imprinting was common in breast cancer we studied 30 patients with sporadic breast carcinoma. A new strategy for detecting intragenic Apa I polymorphism in the exon of IGF2 was used to examine allele-specific expression in the breast cancer specimens by reverse-transcription polymerase chain reaction (RT-PCR). Forty percent (12/30) of the breast cancer samples were identified as heterozygous for IGF2 and studied further. Nine of the 12 heterozygous patients showed biallelic expression of IGF2 by cDNA-PCR, indicating relaxation of normal imprinting at this chromosomal locus. Conclusively, aberrant imprinting of IGF2 in 30% of the breast cancer patients tested provides strong evidence that pathological loss or relaxation of IGF2 imprinting plays an important role in either tumorigenesis or cytokine dysregulation for breast cancer cells.

DNA methylation plays an important role in gene regulation. A human LIM-HOX gene, namely hLH-2, was highly expressed in chronic myelogenous leukemia (CML) and located on chromosome 9q33-34.1, in the same region as the reciprocal translocation that creates the Bcr-Abl chimera of Philadelphia chromosome (H.-K. Wu et al., 1996, Oncogene 12, 1205–1212). To elucidate the mechanism of hLH-2 transcriptional activation, we studied the methylation status of hLH-2 in normal bone marrow and CML cells. When blots containing genomic DNA digested with Hpa II or Msp I were hybridized with full-length cDNA probe, it was discovered that hLH-2 was methylated in normal bone marrow cells in which hLH-2 was not expressed; in contrast, both alleles of the hLH-2 locus in CML cells were heavily hypomethylated. Furthermore, using a sensitive RT-PCR technique, we examined the expression of LH-2 in mouse × human hybrids and a wide array of mouse cell lines containing Abl or Bcr-Abl, and we failed to identify a consistent expression pattern in the cell lines tested. These results suggest that the transcriptional activation of hLH-2 in CML is likely due to a cis -acting effect, but not a trans -acting effect, of the Bcr-Abl fusion protein. Because hypomethylated genes generally are transcribed more efficiently than hypermethylated genes, the high level of hLH-2 mRNA in CML cells probably is a consequence of the low level of methylation of the gene in the leukemic cells.

5098 Background: VHL is a primary cause of inherited RCC and 60% of sporadic clear cell RCCs have mutations in the VHL gene. RCCs often remain asymptomatic for long intervals. Diagnosis during pres...

14687 Background: Recent studies suggest that specific subpopulations of monocytes and myeloid progenitors, particularly those expressing VEGFR-1, play a key role in promoting tumor angiogenesis an...