Detection of low abundance mRNA of myeloid specific genes in cells of acute and chronic lymphoid leukemias by cRNA hybridization

The hybridization to a complementary RNA (cRNA) probe both in situ and in solution was used to assay tiny amounts of mRNA of the lactoferrin (LF) and myeloperoxidase (MPO) genes in normal bone marrow cells and in acute and chronic lymphoid leukemias. Evidence is reported that this technique is much more sensitive than the standard Northern blot technique. The LF mRNA was detectable in three of seven cases of acute lymphoblastic leukemia (ALL) and in three of seven cases of chronic lymphocytic leukemia (CLL). Four cases of ALL were also positive when tested with the MPO cRNA. It is apparent from these results that myeloid specific mRNA, different from MPO, may be detected in leukemic cells with lymphoid phenotype using a method more sensitive than the Northern blot technique. Whether or not the molecular events observed in these cell populations reflect events physiologically occurring rather than a deregulation of gene expression associated to leukemogenesis remains to be established.     

Overexpression of the MPO gene occurring in a case of APL without unusual genotypic characteristics

Northern blot analysis of four typical cases of acute promyelocytic leukemia showed that one of the cell population examined was characterized by a very high level of expression of the myeloperoxidase (MPO) gene. Western blot analysis confirms that the protein content of the cells corresponded to the levels of the MPO mRNA. Southern blot studies of the DNA of this cell population ruled out the presence of any genome amplification or rearrangement. Chromosome hybridization studies in situ confirmed that the MPO gene was translocated on the long arm of chromosome 15. The observation that a typical genomic pattern may or may not be associated with the MPO overexpression leads us to believe that so far it is impossible to reach any conclusion about the significance of the translocation in the genesis of MPO overexpression.     

Expression of the myeloperoxidase gene in acute and chronic myeloid leukemias: relationship to the expression of cell cycle-related genes

The expression of the myeloperoxidase (MPO) gene was studied, by means of Northern blot analysis in 14 cases of acute myeloid leukemia (AML), 11 cases of chronic myeloid leukemia (CML), and 6 cases of CML blast crisis, and in HL60 cells before and after induction of terminal differentiation with retinoic acid (RA), phorbol esters (TPA), or vitamin D. The expression of a panel of cell cycle-related genes, namely C-MYC, histone H3, ornithine decarboxylase, P53, vimentin, and calcyclin, was also studied in the same cell populations. Our results indicate that: (a) MPO gene expression (steady state mRNA levels) is strictly confined to the first stages of myeloid differentiation, reaching its peak at the promyelocyte stage and becoming undetectable in mature granulocytes and monocytes; (b) cells devoid of any detectable MPO enzymatic activity such as leukemic basophils have a high content of MPO mRNA; and (c) MPO gene expression is not related to the growth activity of the cell population. Finally, our results show that the pattern of expression of growth-regulated genes in the neoplastic myeloid disorders AML, CML, and CML blast crisis is remarkably different.     

The localization of the human myeloperoxidase gene is in close proximity to the translocation breakpoint in acute promyelocytic leukemia

The human myeloperoxidase (MPO) gene has recently been cloned in our laboratory. Southern blot hybridization of our MPO cDNA to DNA from a somatic cell hybrid clone panel revealed that the MPO cosegregated with human chromosome 17. In situ hybridization mapped the MPO gene to chromosome 17q22-24. Although this location is close to the translocation breakpoint which occurs in acute promyelocytic leukemia (APL), t(15;17)(q22;q21-22), Southern blot hybridization with different restriction-digested genomic DNA samples from four APL patients did not reveal MPO gene rearrangement. However, RNA dot-blot hybridization showed that APL patients with the translocation expressed high levels of MPO mRNA. This observation raises the possibility that the high levels of MPO gene expression in APL could be due to the arrest of leukemic cells at a specific stage of differentiation or a consequence of the translocation.     

细胞角蛋白基因13在鼻咽癌中的表达研究

目的　探讨细胞角蛋白基因 13(Cytokeratin 13,CK13)在人鼻咽癌 (NPC)中的表达。方法应用免疫组化检测了 40例NPC组织和 8例慢性鼻咽炎 (CINE)组织中CK13蛋白水平的表达 ,同时应用Northern杂交对其中 32例NPC和 8例CINE组织进行了mRNA水平检测 ,并分析了CK13表达与NPC临床特征的关系。结果　在NPC中 ,CK13在蛋白质水平和mRNA水平均较CINE显著降低 (P <0 0 1) ,且CK13表达与NPC颈淋巴结转移有显著相关性 (P <0 0 5 ) ,与肿瘤的T分期无显著相关性(P >0 0 5 )。结论　CK13基因的表达可能与NPC组织分化以及颈淋巴结转移有关。     

Isolated Myeloperoxidase Immunohistochemical Expression in Bone Marrow Biopsy Depicts Clinical Outcomes in Adults with Typical B-Acute Lymphoblastic Leukemia

Introduction: Recently, isolated myeloperoxidase expression (isoMPO) has been documented in B-acute lymphoblastic leukemia (B-ALL) and several contradictory studies addressed its clinical significance in pediatric patients. Aim: In this study, isoMPO was evaluated in bone marrow biopsies (BMB) from adults with B-ALL using immunohistochemistry (IHC) in relation to a number of risk-stratification factors and patients’ outcomes. Methods: Sixty B-ALL adult patients were selected upon electronic database search. Demographic, clinical, laboratory, therapy and survival data were reviewed and tabulated. Flowcytometry (FCM), histopathology and IHC available material were reviewed to confirm the diagnostic criteria according to our standard laboratory protocols. IHC was performed on BMB using antiMPO. Cases were divided into MPO+ve and MPO-ve based on a 3% blast cell staining threshold. Results: Using IHC, 26.7% of B-ALLs were MPO+ve, in most of which ≥10% of blasts were stained. Among standard risk-stratification factors, isoMPO was associated with a mean WBC count above 30x109/L. MPO+ patients achieved therapeutic complete remission at lower rates and were more prone to progressive/refractory disease and relapse. There was a concordant expression of MPO in FCM and IHC. All of the aforementioned parameters reached the level of significance when compared to the MOP-ve group. Kaplan-Meier curves revealed a significantly lower survival probability for the MPO+ group than the MOP-ve one (p= 0.0066; Log-rank test) and also when separating MPO+ and -ve patients by gender (p= 0.0033; Log-rank test). Conclusion: isoMPO occur in a considerable percentage of B-ALL in adults contributing to misdiagnosis. It depicts poor outcomes and might be introduced as a B-ALL risk-stratification factor.     

Amplification and overexpression of the epidermal growth factor receptor gene in human renal-cell carcinoma

We examined 22 cases of renal-cell carcinoma (RCC) for structural alterations of the epidermal growth factor receptor (EGFR) gene and found gene amplification in one case of high-stage-high-grade RCC. Dot blot analysis of the total RNA from tumorous and normal kidney tissues revealed overexpression of the EGFR gene in 12 of 20 (60%) cases of RCC. The highest expression was observed in the gene-amplified case. No correlation was observed between the level of EGFR mRNA and tumor stage or grade. Northern blot analysis revealed normal 10- and 5.6-kb EGFR mRNA bands in RCC. Our data indicate that gene amplification of the EGFR gene is one of the molecular mechanisms of its overexpression in a subset of RCCs.     

Clinical significance of matrix metalloproteinase-7 expression in esophageal carcinoma.

Matrix metalloproteinase-7 (MMP-7) is a member of MMP family and has a wide variety of substrate spectra. It is reported to play an important role in carcinoma invasion and metastasis. There is, however, little information on the clinical significance of MMP-7 in human esophageal carcinoma. We thus studied 48 tumor/normal pair samples of human esophagus by Northern blot analysis. The results demonstrated that the tumor tissue (T) of esophageal carcinoma showed a higher expression of MMP-7 mRNA than the corresponding normal tissue (N) in 31 cases (65%). We also statistically evaluated tumor MMP-7 value (T value) corrected for MMP-7-positive control (KYSE150 transfected with the MMP-7 gene). Fourteen cases with T value > or = 0.3 showed a higher frequency of lymph node metastasis than 34 cases with T value < 0.3 (P < 0.05). The cases with T value > or = 0.3 showed a significantly poorer prognosis than those with T value < 0.3 (P < 0.01). Multivariate analysis demonstrated that the MMP-7 expression status was the independent factor relating to the prognosis (P = 0.0005). The findings indicated that MMP-7 might be a novel prognostic factor for patients with esophageal carcinoma.     

WT1基因在儿童急性B淋巴细胞白血病中的表达及临床意义

目的:探讨WT1(Wilms tumor gene 1)基因在儿童急性B淋巴细胞白血病（B-ALL）中的表达及临床意义。方法:应用实时荧光定量PCR方法检测77例初诊B-ALL患儿WT1基因的相对表达水平,比较患者年龄、性别、流式亚型、染色体核型、临床危险分级等因素的基因表达差异,并随访分析初诊WT1基因表达水平对患儿预后的影响。结果:77例初诊B-ALL患者中,WT1基因阳性率96.10%（74/77）,0~2岁患儿初诊WT1水平高于3~13岁组（P=0.02）,WT1基因表达在性别上无统计学差异（P=0.229）。在对B-ALL流式亚型间WT1的分析中显示,Pro-B-ALL患者表达量最高,Pre-B-ALL次之,Com-B-ALL最低（两两比较差异,P值分别为0.002,0.008,0.040）。临床高危（P=0.041）、t(4;11）（P=0.034）、初诊白细胞大于50×109/L（P=0.009）的患者有更高的WT1表达。首次诱导化疗后细胞形态学缓解与否,其初诊时WT1表达无明显差异（P=0.84）,但长期随访得到持续缓解的患者初诊WT1表达低于复发或不缓解的患者。结论:WT1基因在儿童B-ALL中表达的高低可能提示患者白血病细胞的分化水平,并与长期预后相关。     

EXPRESSION OF THE MULTIDRUG RESISTANCE GENE IN LEUKEMIC CELLS

P-glycoprotein(Pgp; Mr=170,000) to encoded by a family of genes-Multldrug resistance gene. The Pgp has been demonstrated to mediate resistance to multiple structurally dissimilar drugs, which fuctions as an energy- dependent efflux pump so that a cell with high level of mdr expression can more effectively eliminate cytotoxic drugs. In this report, a simplified method for analysis of clinical samples and assess the level of gene expression was set up. Furthermore, by using 32P labelled mdr- 1 cDNA as the probe and RNA dot blotting the mdr-mRNAs from 5 cases of myeloblastic leukemia cells were analysed. It was shown that the level of mdr-1 expression In different myeloleukeic cells was various and reduced In one case after remission. The established method for mRNA analysis could be generalized for evaluating the level of mRNA in clinical samples.     

Vascular endothelial growth factor overexpression is correlated with von Hippel-Lindau tumor suppressor gene inactivation in patients with sporadic renal cell carcinoma

BACKGROUND: A high frequency of genetic alterations of the von Hippel-Lindau (VHL) gene and overexpression of the vascular endothelial growth factor (VEGF) gene have been observed independently in human sporadic renal cell carcinoma (RCCs), but to the authors' knowledge the association between the two has not been characterized in primary sporadic RCC. In the current study the authors report the simultaneous comparison of the biallelic inactivation status of the VHL gene and VEGF expression levels in patients with sporadic RCC. METHODS: DNA and RNA were extracted from 27 sporadic RCC samples. Mutation was analyzed by direct sequencing of the amplified VHL DNA and cDNA. Loss of heterozygosity (LOH) of the gene was analyzed at three polymorphic markers. The VEGF mRNA was measured using Northern blot analysis. RESULTS: Mutations of the VHL gene were found in 14 of 27 RCC samples (51.9%). LOH analysis by a VHL-intragenic polymorphic marker and 2 extragenic microsatellite markers, D3S1560 and D3S1317, showed that LOH occurred in 10 of 15 RCC samples (66.7%). Overexpression of VEGF mRNA was observed in 17 of 27 RCC cases (63.0%): 15 of the 18 RCC samples estimated to have at least 1 hit, but only 2 of the 6 RCC samples with 0-1 hit, and none of the 3 RCC samples in which the VHL gene was not inactivated. CONCLUSIONS: VEGF overexpression was found to be correlated with both monoallelic and biallelic VHL inactivation. Alteration of the VHL gene is believed to cause angiogenesis in RCC cases through the overexpression of VEGF.     

Expression of collagenase-related metalloproteinase genes in human lung or head and neck tumours.

We address the question as to whether increased metalloproteinase production might be related to the high regional recurrence rate of some carcinomas, and particularly head and neck squamous-cell carcinomas (SCC). Northern blot of total RNA prepared from 26 lung carcinomas, 107 head and neck carcinoma samples and corresponding normal tissue samples demonstrates the frequent and sometimes concomitant over-expression of the 2 stromelysin genes, the type-I collagenase gene and the pump-I gene in the head and neck tumour tissue samples. In these SCC, over-expression of the 2 stromelysin genes and the type-I collagenase gene (but not the pump-I gene) is associated with a high degree of tumour differentiation. Moreover, a tumour with high levels of the stromelysin mRNAs is more likely to show high local invasiveness, suggesting that the stromelysins may be implicated in the clinical course of head and neck tumours. Evaluation of the corresponding mRNA levels may prove a useful indicator for predicting the clinical aggressiveness of these tumours.     

Clinical significance of secreted protein acidic and rich in cystein in esophageal carcinoma and its relation to carcinoma progression

BACKGROUND: Secreted protein acidic and rich in cystein (SPARC) is a small extramatrix-associated protein. Its production increases during angiogenesis and enhances matrix metalloproteinase-2 (MMP-2) expression. The goal of this study was to show the clinical relevance of SPARC and its relation to MMP-2 expression in esophageal carcinoma patients. METHODS: The authors investigated SPARC mRNA expression in 48 tissue samples of esophageal tumors characterized by MMP-2 mRNA expression in a Northern blot analysis. Western blot analysis and immunohistochemistry were also performed in esophageal carcinoma tissue samples. RESULTS: All 48 tissue specimens had high expression of SPARC mRNA. Quantitative evaluation showed that high SPARC mRNA was associated significantly with lymph node metastasis (P = 0.05) and poorer prognosis (P = 0.025). Expression of SPARC mRNA was associated significantly with MMP-2 mRNA expression (R = 0.65; P < 0.01). Both SPARC and MMP-2 were immunolocalized intensely in carcinoma and stromal cells, whereas normal esophageal mucosa and submucosa did not express SPARC. The 35-kilodalton cleaved SPARC was detected in esophageal carcinoma tissue specimens by Western blot analysis and it was associated with MMP-2 mRNA expression. CONCLUSIONS: In terms of clinical significance, SPARC accumulation may reflect a functional correlation with MMP-2 and the associated expression could play a key role in the progression of esophageal carcinoma.     

C－Ha－ras癌基因高表达与胃癌临床行为的关系

应用ＲＮＡ斑点杂交、Ｎｏｒｔｈｅｒｎｂｌｏｔ和免疫组织化学技术检测了２０例胃癌病员癌组织中ｃ－Ｈａ－ｒａｓｍＲＮＡ和Ｐ２１蛋白的表达。统计学结果表明，胃癌组织中ｃ－Ｈａ－ｒａｓｍＲＮＡ过度表达和肿瘤淋巴结转移状态密切相关（Ｐ＝０．０３３），随着胃癌临床病程进展，肿瘤细胞中ｃ－Ｈａ－ｒａｓＰ２１表达水平逐渐升高（Ｐ＝０．１１２）。提示ｃ－Ｈａ－ｒａｓ癌基因高表达可能是胃癌形成过程中的关键步骤，ｃ－Ｈａ－ｒａｓ癌基因产物可作为胃癌新的生物学标志来判定肿瘤的预后并指导临床治疗。     

急性淋巴细胞白血病患者 ASB2和 Jak3基因mRNA 表达的研究

目的：研究急性淋巴细胞白血病（ALL）患者骨髓中锚蛋白重复序列和抑制细胞因子信号盒蛋白2（ASB2）和 Janus 激酶3（Jak3）mRNA 的表达及其两者的相关性。方法收集初诊的48例 ALL患者（37例 B 细胞 ALL,11例 T 细胞 ALL）和34例非白血病患者（对照组）骨髓,采用实时荧光定量 PCR检测骨髓中 ASB2和 Jak3 mRNA 表达情况。结果B 细胞 ALL 和 T 细胞 ALL 患者骨髓中 ASB2 mRNA表达量相对于对照组分别升高了32．7倍和68．5倍,差异均有统计学意义（t ＝20．1,P ＜0．01;t ＝23．1, P ＜0．01）,Jak3 mRNA 表达量较对照组分别升高了2336．3和7131．5倍（t ＝70．2,P ＜0．01;t ＝90．4, P ＜0．01）。ASB2和 Jak3 mRNA 表达量具有相关性（r ＝0．523,P ＜0．001）。结论ASB2和 Jak3在 ALL患者骨髓中异常表达,且具有正相关性,两者可能共同参与白血病细胞的恶性增殖和异常分化。     

Early expression of glycophorin C during normal and leukemic human erythroid differentiation

Glycophorins C and D (GPC and GPD) are two erythrocyte glycoproteins which originate from the same gene but differ in their NH2-terminal residues. The cell surface expression of these glycoproteins during normal and erythroid differentiation has been investigated with monoclonal and polyclonal antibodies and has been compared to the expression of glycophorin A (GPA), the major sialoglycoprotein of human red cells. Using glycosylation-independent antibodies (monoclonal or polyclonal), GPC or GPD was detected in erythroid and nonerythroid cell lineages. However, a glycosylation-dependent monoclonal antibody (MR4-130) detected an epitope on GPC which appears to be erythroid specific, suggesting that lineage specificity of this glycoprotein is related to some carbohydrate structures. During normal erythroid differentiation, GPC was expressed early at the level of erythroid progenitors (part of erythroid burst-forming unit and erythroid colony-forming unit) as detected with a glycosylation-independent monoclonal antibody (APO 3), whereas GPA is only present during terminal erythroid differentiation. The MR4-130 epitope was not coordinately expressed on the cell surface with the GPC molecule in the erythroid differentiation, since it was detected at the level of the more mature erythroid colony-forming unit slightly later than the GPC polypeptide. In four erythroleukemic patients, blast cells blocked at discrete stages of the erythroid differentiation were also investigated with antibodies and complementary DNA probes for GPA and GPC. GPA was immunologically detected in three of four cases, and its cell surface expression was correlated with the amount of specific mRNA in the cells, as seen by Northern blot analysis. GPC was immunologically detected on the blast cells of all four patients. However, in two cases including one with positive expression of GPA, the MR4-130 epitope was absent from the GPC molecule. By Northern blot analysis, we found that the GPC/GPD mRNA was present at a high level in all four patient samples. Western blot analysis of GPC and GPD in two of these patients revealed that these mRNAs were mostly translated into the GPD molecule, suggesting that these glycoproteins might be differently processed in certain cases of erythroleukemia.