Polyamine profiles and growth properties of ornithine decarboxylase overexpressing MCF-7 breast cancer cells in culture

Summary To determine the direct influence of the polyamine (PA) pathway on breast cancer phenotype, we employed a transfection approach to induce overexpression of the PA biosynthetic enzyme ornithine decarboxylase (ODC) in the hormone-responsive MCF-7 breast cancer cell line. Using a modified calcium phosphate method and an ODC cDNA coding for a truncated and more stable enzyme, we were able to achieve a moderate to marked degree of ODC overexpression (up to 150-fold) in a transient transfection system. ODC-overexpressing MCF-7 cells exhibited a selective increase in cellular putrescine content, while the levels of spermidine and spermine remained unaffected. Under defined culture conditions, overexpression of ODC resulted in a consistent but modest increase in [³H]thymidine incorporation into DNA which was similar in the presence and absence of 17--estradiol, TGF-, and IGF-I. In the presence of serum, the effect of ODC overexpression on basal [³H]-thymidine incorporation into DNA was inconsistent, possibly as a result of subtle differences in culture conditions. Overall, our results support the hypothesis that activation of the PA biosynthetic pathway may confer a growth advantage to breast cancer cells.     

Effects of exogenous agmatine in human leukemia HMC-1 and HL-60 cells on proliferation, polyamine metabolism and cell cycle

Impairment of agmatine homeostasis is involved in the regulation of cell proliferation in malignant solid tumors. The present study aimed at analyzing the relevance of agmatine homeostasis in pathophysiology of human leukemia. Proliferation of the human leukemia cells HMC-1 and HL-60 was determined in the absence or presence of agmatine. Apoptosis and cell cycle distribution was investigated by determination of caspase-3 activity and/or flow cytometry after staining with propidium iodide. Expression analysis was performed by qPCR and by a microarray genechip. Exogenous agmatine inhibited proliferation of both HMC-1 and HL-60 cells. The antiproliferative effect was due to interference of agmatine with the cell cycle with no evident signs of apoptosis. Comparative analysis of expression of mRNA in untreated HMC-1 cells and in non-leukemic human mast cells revealed a much lower expression of agmatinase and diamine oxidase in HMC-1 cells indicating a significantly reduced agmatine catabolism in the leukemic cells. At the mRNA level, inhibition of proliferation of both cell lines by agmatine was associated with cell type-specific alterations of the expression of enzymes of the polyamine pathway. The present results point to a significant role of agmatine homeostasis in the (patho)physiology of cell proliferation of leukemic cells, at least in HMC-1 and HL-60 cells, that may serve as a potential target for an adjuvant therapy in the treatment of human leukemia.     

鸟氨酸脱羧酶和S-甲硫氨酸脱羧酶双反义RNA腺病毒抑制食管癌细胞的增殖和侵袭

目的:探讨鸟氨酸脱羧酶(ornithine decarboxylase,ODC) 和 S-甲硫氨酸脱羧酶(s-adenosylmethionine decarboxylase,AdoMetDC)的双反义RNA腺病毒载体(Ad-ODC-AdoMetDCas)对食管癌Eca109细胞增殖和侵袭的抑制作用.方法:重组腺病毒载体Ad-ODC-AdoMetDCas感染食管癌Eca109细胞,应用Western blotting和HPLC分别检测双反义RNA腺病毒对食管癌细胞中ODC和AdoMetDC蛋白表达以及胞内多胺合成的抑制作用.采用活细胞计数法观察其对食管癌Eca109细胞生长增殖的影响,采用Matrigel侵袭实验检测重组腺病毒载体感染对食管癌Eca109细胞侵袭活性的影响.同时应用裸鼠皮下移植瘤模型观察Ad-ODC-AdoMetDCas对移植食管癌生长增殖的抑制作用.结果:Western bloting证实Ad-ODC-AdoMetDCas可明显抑制食管癌Eca109细胞中ODC和AdoMetDC蛋白的表达,HPLC结果显示食管癌Eca109细胞感染Ad-ODC-AdoMetDCas后细胞内腐胺、精胺、精脒等3种多胺含量都明显降低(P<0.01).活细胞计数法表明Ad-ODC-AdoMetDCas对食管癌Eca109细胞生长增殖有明显抑制作用(P<0.01),Matrigel侵袭实验结果显示Ad-ODC-AdoMetDCas可显著降低食管癌Eca109细胞的体外侵袭能力(P<0.01).体内实验显示,双反义RNA腺病毒载体对已形成的裸鼠皮下移植瘤具有明显的抑制作用(P<0.05或P<0.01).结论:ODC和AdoMetDC双反义RNA重组腺病毒能显著抑制食管癌细胞的增殖和侵袭,具有食管癌基因治疗临床应用的潜在价值.     

Estradiol control of ornithine decarboxylase mRNA,enzyme activity,and polyamine levels in MCF-7 breast cancer cells: therapeutic implications

Previous studies have shown that natural polyamines - putrescine, spermidine, and spermine - play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G₁ synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G₁ phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H₂N(CH₂)_nNH(CH₂)₃NH₂ (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 µM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [³H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.     

T1 and T2 Proton Nuclear Magnetic Resonance (N.M.R.) relaxation times in vitro and human intracranial tumours

Summary 137 samples of intracranial tumours have been studied in proton NMR spectroscopy. T1 and T2 relaxation times are above those of normal grey and white matter. Differential diagnosis between benign and malignant brain tumours does not seem feasible upon proton T1 and T2 alone. Histological correlations allowed us to specify secondary changes accounting for T1 and T2 variations (oedema, microcyst, stroma reaction, necrosis).     

Clusterin (SGP-2, ApoJ) expression is downregulated in low- and high-grade human prostate cancer

Clusterin is overexpressed during tissue and cell involution and downregulated in proliferating cells. Its role in cell survival, cell death and neoplastic transformation remains debated. We studied the expression and distribution of clusterin mRNA and protein in human prostate carcinoma (CaP) specimens of different degrees of malignancy. Fresh CaP specimens were obtained from 25 patients subjected to long-term androgen ablation before surgery. Clusterin expression was studied by Northern and Western analysis, in situ hybridization and immunohistochemistry, in comparison with Gas1 and histone H3 mRNA (markers of cell quiescence and S phase of the cell cycle, respectively). Clusterin is downregulated in CaP in comparison with matched benign controls. In low-grade CaP, clusterin colocalized with Gas1 to the stromal compartment, and in some glands, the basal lamina was heavily stained. In high-grade CaP clusterin stained the remnants of stromal matrix while histone H3 localized to cancer cells, which were very rarely clusterin positive. High clusterin expression was found in the branches of a nerve infiltrated by tumor. The periglandular clusterin expression found in low-grade CaP could result from induction of quiescence and/or apoptosis of prostatic fibroblasts lining those glands in which tumor invasion is at an initial stage, involving basal lamina. In advanced CaP, the staining of the remnants of the extracellular matrix suggests a role for clusterin in the process of dismantling the stromal organization caused by cancer progression.     

Metabolic patterns in malignant gliomas

Summary Changes of mitochondrial and cytoplasm tumor metabolism were studied in malignant gliomas and normal cortex probesin vitro. By spectrophotometric methods marker enzymes of different mitochondrial (whole respiratory chain, citrate acid cycle, fatty oxidation) and cytoplasm (glycolysis, pentose phosphate shunt) metabolic energy pathways were analysed. Generally, the activities of intramitochondrial key enzymes were significantly decreased in gliomas when compared with enzyme activities of normal cortex tissue (p < 0.01). Glycolytic enzymes and a representative of the pentose phosphate shunt were unchanged or increased. Ratios of marker enzymes of the glycolytic pathway (lactate dehydrogenase) and glycose-6-P dehydrogenase revealed a significant difference between glioblastomas (p < 0.05) and grade III (p < 0.05) tumors in comparison to normal astrocytic tissue and astrocytomas WHO grade II. Thus, biochemical analyses allow metabolic grading of gliomasin vitro and may be a useful tool for understanding tumor biology.     

Chlorpheniramine inhibits the synthesis of ornithine decarboxylase and the proliferation of human breast cancer cell lines

Summary Proliferation of both mouse and human breast cancer cells was inhibited by chlorpheniramine (CPA) in a dose-response manner. At the beginning of the exponential phase of growth (two days after seeding), 250 µM CPA was able to reduce cell proliferation by 75% (in Ehrlich cell cultures) and 30% (in MCF-7 cultures). The antiproliferative effect of CPA was also tested on a poorly-differentiated and hormone-insensitive human breast cancer cell line (MDA-MB231) and on a highly proliferative human colon cancer cell line (clone 3). CPA was cytotoxic for MDA-MB231 cells at concentrations higher than 50 µM, and it was also cytotoxic for the colon cancer cell clone 3 at 250 µM CPA. Nevertheless, colon cancer cells were slightly stimulated at CPA concentrations less than 100 µM. CPA reduced (by 50–70%) the ornithine decarboxylase induction occurring early after culture seeding of experimental mammary tumors (Ehrlich carcinoma cells) and human breast cancer cells (MCF-7). The presented data suggest that in addition to ODC inhibition, CPA presents other still unknown cytotoxic effects.     

A correlative study of gliomas usingIn vivo bromodeoxyuridine labeling index and computer-aided malignancy grading

An in vivo bromodeoxyuridine (BrdU) labeling index (LI) was estimated in 43 cases of astrocytic tumors and mixed gliomas by one hour intra-operative intravenous infusion at a dose of 200 mg/m2 and correlated with (a) histological grading using a computer aided malignancy classifier TESTAST-268; and (b) histological typing using WHO classification. The lowest BrdU LI was seen in pilocytic and gemistocytic astrocytomas followed by astrocytomas, anaplastic astrocytomas and glioblastoma multiforme in that order. Mixed oligoastrocytomas followed the pattern of their astrocytic counterparts. Tumors of similar histological type showed different BrdU LI values especially amongst astrocytomas and glioblastomas. A statistically significant difference in the BrdU LI was also noted between the higher TESTAST grades of astrocytomas (T III and IV) versus the lower TESTAST grades (T II). Unlike earlier reports in literature, in the present study the category of BrdU LI of <1 contained no case of anaplastic astrocytoma or glioblastoma multiforme (TESTAST grades III and IV). Likewise, the category of BrdU LI >5 contained only anaplastic astrocytoma and glioblastoma multiforme (TESTAST grades III and IV). Maximum spread of cases was seen in the BrdU LI category of 1-5, not only in terms of histological types but also TESTAST grades. Thus there appeared to be a positive trend of increasing BrdU LI values both with histological types and increasing TESTAST grades. Further, an interesting observation was that by using a combination of TESTAST grades and BrdU LI, the histologically homogenous glioblastoma group could be further subdivided into 4 categories which showed a trend towards prognostic correlation. Thus, this study though preliminary with number of cases being small in some groups, highlights the possible usefulness of combined histological typing, TESTAST grading and in vivo BrdU LI for prognostication of gliomas especially glioblastoma multiforme.     

X刀治疗脑胶质瘤的方法及结果分析

本文报告了我院从1995年3月到1996年5月，采用德国BrainScanX刀治疗系统治疗脑胶质瘤病人45例，经过6～12个月的随访，结果表明，总的治疗有效率达到75．56％，半年以上的生存率为77．78％，颅内压增高等严重并发症的发生率为22．22％。作者总结了适应症选择的标准、治疗计划设计优化的原则、疗效判断标准、X刀后病理和影像变化的特声、以及与并发症发生有关因素等方面的经验和体会。     

Treatment of infants with malignant gliomas: The Pediatric Oncology Group Experience

Although survivals of infants with malignant brain tumors are worse than any other age group, one possible exception to this rule are the malignant gliomas. Eighteen children less than 3 years of age with malignant gliomas (glioblastoma multiforme, anaplastic astrocytoma and malignant glioma) were treated on the Pediatric Oncology Group regimen of prolonged postoperative chemotherapy and delayed irradiation, (1986–1990). Of 10 children evaluable for neuroradiologic response, 6 had partial responses (> 50% reduction) to two cycles of cyclophosphamide and vincristine. Progression free survivals at l, 3 and 5 years were 54.25% ± 12, 43% ± 16 and 43% ± 23 respectively. Survivals at 5 years were 50% ± 14. Four children were not irradiated after 24 months of chemotherapy due to parental refusal and none have developed recurrent disease. Neither degree of surgical resection, presence or absence of metastases, nor pathology influenced survival but this may reflect small sample size. This study suggests that some malignant gliomas in infants are chemotherapy sensitive and may be associated with a good prognosis. Why infants with these high-grade gliomas fare better than adults is not clear. It is likely that there is something intrinsically different about them that cannot be identified on routine pathologic examination.     

Postoperative radiotherapy and radiotherapy combined with CCNU chemotherapy for treatment of brain gliomas

A prospective, randomized trial evaluates the effects of two postoperative treatment regimens on survival in 198 adult patients with supratentorial gliomas. All patients were irradiated with 6 000 rads after possibly radical removal of tumors. CCNU administration in the dosis of 100 mg/sq m of body surface every 6–8 weeks following surgery proved to have no significant effect on the survival of patients. The median survival time in patients receiving radiation therapy alone was 61±7 weeks, while in those receiving additional chemotherapy was 56±4 weeks. Tumor histological malignancy and patients age were found to be the only important prognostic factors, irrespective of the treatment modality. Address for offprints: T Trojanowski, Department of Neurosurgery, Medical School, Jaczewskiego 8, 20-950 Lublin, Poland     

Arachidonic acid metabolic profiles in human meningiomas and gliomas

We determined arachidonic acid (AA) cyclooxygenase metabolic profiles in specimens of human intracranial tumors (gliomas and meningiomas) and, when available, normal brain tissue. Samples were collected at surgery and immediately frozen in liquid nitrogen. The five stable metabolites of AA (PGE2, PGD2, PGF2, 6-keto-PGF1 and TXB2) were measured by high-resolution gas chromatography-mass spectrometry after ex vivo metabolism of endogenous AA by tissue homogenates. The absolute amounts of AA metabolites varied widely between samples, though meningiomas and gliomas showed characteristic profiles. Compared to the slow-growing benign meningiomas, the rapidly-growing infiltrating gliomas had higher synthesis of TXA2 (reported as a procancer metabolite) and lower synthesis of PGD2 and PGI2 (reported as anticancer metabolites). A higher overall synthesis capacity, preferentially toward TXA2, was found in glioblastomas than in nonpathological brain tissue.This paper was presented in part at the Sixth International Conference on Brain Tumor Research and Therapy, Asheville, North Carolina, October 20–23, 1985.     

Red blood cell polyamines as a diagnostic indicator of glioma presence and recurrence

Summary Red blood cell (RBC) polyamines, spermine and spermidine have been assayed from 103 patients with confirmed intracranial tumours and 87 controls. For spermine the test had a specificity of 97% and a sensitivity of 30% in preoperative patients, increasing to 40% in glioma patients. Tumour recurrence in postoperative patients gave a sensitivity of 48% increasing to 60% in glioma groups. For initial tumour presentation, therefore, this test is suggestive, though not unequivocal, of tumour presence. Its major role probably lies in monitoring the progress of the patient and its major importance in the detection of tumour recurrence.     

Localized proton spectroscopy of inoperable brain gliomas. Response to radiation therapy

Within vivo 1H-MRS resonances of several metabolites were simultaneously measured in cerebral gliomas and adjacent normal brain. 15 patients with inoperable brain gliomas all histologically verified were monitored with 1H-MRS and MRI before and after radiotherapy. 11 patients were evaluable. 1H-MRS technique evolved from single volume measurements to one dimensional and two dimensional 1H spectroscopic imaging. In all patients N-acetyl-aspartate signals were decreased in tumour areas compared to the normal brain hemisphere. No recovery was seen after radiotherapy. Choline signals were increased in tumour margins of high grade gliomas and more diffusely in low grade gliomas. In 5 patients the choline resonance decreased after radiotherapy, accompanied by a shrinkage of tumour diameter on MRI. Lactate signals were present in high grade and unspecified astrocytomas and absent in most low grade gliomas. In 3 patients the lactate signal disappeared after radiotherapy. These observations indicate the feasibility of 1H-MRS in monitoring metabolic responses on radiotherapy of brain gliomas.