Tumour necrosis factor‐α and interleukin‐1 and ‐6 in fibrocystic breast disease

The risk of developing breast cancer is higher in women presenting gross cystic disease (cysts > 3mm in diameter) of the breast with intracystic K+/Na+ > 3 as compared with K+/Na+ < 3. The present study reports the levels of tumour necrosis factor (TNF), interleukin1 (IL1), and interleukin6 (IL6) in the breast cyst fluid of women with gross cystic disease and analyses the relationship between the intracystic concentration of these cytokines, sex steroid hormones, and the K+/Na+ ratio. The concentration of these cytokines, estradiol, testosterone, dehydroepiandrosterone sulfate (DHEAS), and 17OHprogesterone were determined in the breast cyst fluid of 54 women with gross cystic disease. No significant differences were found in the cystic levels of IL1 between cysts with intracystic K+/Na+ < 3 and > 3. However, in cysts with intracystic K+/Na+ > 3 we found a lower concentration of IL6 and TNF than in those with intracystic K+/Na+ < 3.Stepwise multiple linear regression analysis demonstrated that the concentration of IL6 in breast cyst fluid was predicted statistically by a negative regression coefficient for the concentration of estradiol and DHEAS, and by a positive regression coefficient for the concentration of TNF. The concentration of TNF in breast cyst fluid was predicted statistically by a positive regression coefficient for the concentration of IL6, and by a negative regression coefficient for the concentration of estradiol. No candidate variable was included in the model to predict concentrations of IL1 in breast cyst fluid. Our results indicate that IL6 and TNF could have a local protector role in gross cystic disease, and that they could be used as a marker to identify cyst type.     

Hereditary persistence of {alpha}-fetoprotein: Case report and review of the literature

Persistently elevated -fetoprotein (AFP) levels of 24 to 30µg/ml (normal <10 µg/ml) were found in a 38-year-old healthyman. Subsequently, AFP was found to be elevated in another five out of 13family members within three generations. The pedigree is consistent with anautosomal dominant inheritance pattern. No discernible disease and nofunctional abnormality appears to be associated with this clinically benigndisorder which has been recorded in the literature on four occasions todate. The reported AFP levels in these other cases ranged from 18 to 198µg/ml.Physiologically, AFP is mainly produced in the liver and the yolk sac ofhuman fetuses more than four weeks old, with peak values of up to 4 mg/ml at12 to 16 weeks of gestation. After birth, AFP levels usually fall, withineight to 12 months, to a very low concentration of <10 µg/ml andpersist at low levels throughout life. However, AFP levels can rise abovenormal in both children and adults in distinct conditions and diseases whichwill be discussed.Hereditary persistence of -fetoprotein (HPAFP) should be consideredin both children and adults with unexplained and persistent elevation of AFPe.g., those screened for hepatocellular carcinoma or diagnosed for germ celltumor. It should also be recognized in AFP screening for neural tube defectsor Down's syndrome during pregnancy. Hereditary persistence of AFP can beeasily confirmed by analyzing AFP levels in family members.     

Prognostic relevance of transforming growth factor alpha (TGF-α) and tumor necrosis factor alpha (TNF-α) detected in breast cancer tissues by immunohistochemistry

Summary The function of different growth factors in the development and progression of malignant tumors and the role of cytotoxic cytokines in the host response generated against neoplasms have been recently studied. Anti-TGF- and anti-TNF- monoclonal antibody families have been developed and characterized previously by our laboratory. Libraries of anti-TGF- and anti-TNF- monoclonal antibodies were selected for equal immunoreactivity both in native (frozen) and in formaldehyde fixed, paraffin embedded histological sections. No differences were found between native and fixed samples demonstrated in 10 cases in the present prospective study. Retrospective investigation was performed in 35 histopathological specimens of breast cancer patients detailed clinically and observed during 5 years after the surgical treatment. Correlation between TGF- and/or TNF- expression and clinical staging - TNM score, lymph node metastasis, tumor recurrence and survival time - was analyzed. According to our present study, the TGF- positive patients had worse clinical prognosis than the TNF- positive and double positive cases during long term observation.     

Organ specificity of tumor metastasis: role of preferential adhesion,invasion and growth of malignant cells at specific secondary sites

The locations of distant secondary tumors in many clinical cancers and animal tumors are nonrandom, and their distributions cannot be explained by simple anatomical or mechanical hypotheses based on the simple lodgment or trapping of tumor cell emboli in the first capillary bed encountered. Evidence from certain experimental tumor systems supports Paget's seed and soil hypothesis on the nonrandom distributions of metastases, in which the unique properties of particular tumor cells (seeds) and the different characteristics of each organ microenvironment (soil) collectively determine the organ preference of metastasis. Experimentally, differential tumor cell adhesion to organ-derived microvessel endothelial cells and organ parenchymal cells, differential invasion of basement membranes and organ tissues, and differential responses to organ-derived growth-stimulatory and-inhibitory factors all appear to be important determinants in explaining the organ preference of metastasis. Each tumor system may achieve organ specificity because of its own unique set of multiple metastasis-associated properties and responses to host microenvironments. As neoplasms progress to more highly malignant states multisite metastases are more likely and organ-specific metastases may be masked or circumvented owing to stochastic events, tumor cell diversification, host selection processes, and increased production of tumor autocrine molecules that may modulate adhesion, invasion, growth, and other properties important in metastasis. The importance of each of these properties, however, appears to vary considerably among different metastatic tumor systems. These and other tumor cell and host properties may eventually be used to predict and explain the unique metastastic distributions to certain human malignancies.     

Evaluation of response to postradiation eight in one chemotherapy in childhood brain tumors

Ten children, 3 to 15 years of age with high risk primary brain tumors were treated with postradiation eight in one chemotherapy; vincristine, lomustine, procarbazine, hydroxyurea, cisplatin, cytosine arabinoside, cyclophosphamide and methylprednisolone. The tumors comprised of three medulloblastomas, two primitive neuroectodermal tumors, one ependymoblastoma and four anaplastic ependymomas. Treatment involved surgery (two total resection, six subtotal and two biopsy only) followed by conventional radiotherapy (primary tumor: 50–54 Gy, whole brain: 30–45 Gy, and spinal axis: 25–36 Gy). Objective tumor response with radiotherapy was achieved in 7 of 9 patients (78%) (6/8 patients with residual tumor and one patient with complete resection but positive cerebrospinal fluid cytology). Complete response was attained in 4 of 9 patients (44%). Eight in one chemotherapy was initiated four weeks after radiation and repeated at 4 weekly intervals for 5–8 courses. Postradiation eight in one failed to show any additional effect on tumor responses. Median survival was 34 months (range 9–48 months) with five of ten patients alive: four in complete and one in partial remission. All the five survivors were among the patients who had achieved response to initial treatment. This result suggested that degree of response to initial treatment might determine subsequent outcome and thus the choice of modality for initial therapy might be important.     

The effects of TGF-α and 17β-estradiol on polyphosphoinositide metabolism in MCF-7 breast cancer cells

Summary The mechanism by which transforming growth factor-alpha (TGF-) stimulates breast cancer cell proliferation is largely unknown. Furthermore, its potential role as an autocrine effector of estradiol-17 (E₂)-stimulated growth of hormone-dependent mammary tumors remains controversial. Transient changes in phosphatidylinositol (PI) turnover have been demonstrated in several tissues in response to growth factors. In these experiments, we tested the effects of TGF- and E₂ on PI metabolism in three MCF-7 breast cancer cell sublines (MCF-7B, MCF-7I, and MCF-7J). Although TGF- was mitogenic in MCF-7I and MCF-7J cells, PI hydrolysis was stimulated by the growth factor only in the MCF-7I cells. In addition, the TGF- effect was relatively modest, ranging from 23% to 42%. E₂ effects on PI turnover were tested in the MCF-7B cells, which were the most sensitive to the proliferative effect of the hormone. E₂ did not stimulate PI hydrolysis, whether or not the cells were labelled in the presence of the hormone. On the other hand, E₂ did seem to stimulatede novo synthesis of phosphatidylinositol and induce activation of PI kinases. These results demonstrate that TGF--stimulated PI hydrolysis is modest and cell type dependent. At least under certain conditions, PI metabolism is not involved in the proliferative effects of TGF- (MCF-7J) or E₂ (MCF-7B). The role of increased PI synthesis in E₂-stimulated MCF-7 cell growth remains to be established.This work is supported by a grant from the National Cancer Institute, POI CA40011.     

Expression of TGFα in Meningiomas

The objective of this study was to examine the expression of transforming growth factor (TGF), a mitogen for many cell types, and its receptor in basic subtypes of meningiomas as well as in meningiomas of varying grade. Formalin-fixed tissues from 26 meningiomas including 15 benign (5 meningothelial, 5 transitional, and 5 fibrous variants), 6 atypical, and 5 malignant examples were immunohistochemically examined for both TGF protein and EGF/TGF receptor protein. In addition, in situ hybridization (ISH) was used to detect TGF mRNA expression. Immunostaining for TGF was strongest in fibrous and atypical meningiomas, followed closely by transitional and malignant tumors. Only weak reactivity was observed in the meningothelial variant. In all but 4 tumors (2 fibrous, 2 atypical), ISH showed TGF mRNA to be present, the signal being stronger in malignant than in conventional or atypical tumors. Lastly, immunostaining for EGF/TGF receptor was positive in all tumors studied. Strong TGF protein expression in meningiomas is commonly associated with fibrous morphology. Although the frequent detection of both TGF protein and its mRNA, as well as of EGF/TGF receptor within tumors of all type and grades, suggests that TGF serves to promote tumor growth, its possible role in tumorigenesis or malignant progression is uncertain.In summary, demonstration of these substances is of no utility in the classification or grading of this common tumor because the differences in their expression among the various meningioma subtypes were not statistically significant.     

Increased Risk of Developing Hepatocellular Carcinoma Associated with Carriage of the TNF2 Allele of the −308 Tumor Necrosis Factor-α Promoter Gene

BACKGROUND AND AIMS: Tumor necrosis factor-alpha (TNF-alpha) is a cytokine that may act as an endogenous tumor promoter. A genetic polymorphism of TNF-alpha at position -308 of the promoter region, which includes TNF1 (-308G) and TNF2 (-308A) alleles, has been found to be associated with susceptibility to various types of cancer. We conducted a study to evaluate the association between this polymorphism and hepatocellular carcinoma (HCC). METHODS: We recruited 74 HCC patients and 289 healthy controls, and determined their -308 TNF-alpha promoter genotypes through polymerase chain reaction followed by electrophoresis. RESULTS: Carriage of the TNF2 allele was associated with an increased risk of HCC (odds ratio [OR] = 3.5; 95% confidence interval [CI]:[2.1, 6.0]), and a trend toward a significant increase in the risk of developing HCC was observed from TNF1/TNF1, TNF1/TNF2, to TNF2/TNF2 genotypes (p < 0.01). After adjustment for gender, age, and markers of hepatitis B and C, the OR of developing HCC associated with TNF2 allele carriage was 5.3 (95% CI: [2.3, 12.1]; p < 0.01) CONCLUSIONS: Carriage of the TNF2 allele is a significant predictor of HCC independent of hepatitis B and C, and therefore it may be used as a biomarker for susceptibility to HCC.     

Interferon effect on glycosaminoglycans in mouse gliomain vitro

Summary The effect of mouse interferon / (MuIFN /) on the production of glycosaminoglycans (GAGs) by mouse glioma G-26in vitro was evaluated. Two GAG species secreted extracellularly by the mouse glioma G-26 were isolated using cellulose acetate electrophoresis. They were identified as hyaluronic acid (HA) and chondroitin sulfate (CS) following enzymatic digestion with enzymes: hyaluronidase and chondroitinase ABC. Further characterization of CS by enzymatic digestion with specific chondroitinases for chondroitin 4-sulfate (CSA) and chondroitin 6-sulfate (CSC), revealed that the isolated CS was neither CSA nor CSC. Therefore, it may be either chondroitin sulfate B (CSB) (dermatan sulfate) or one of the chontroitin sulfate isomers (D-H).The three day incubation of glioma G-26 cells with 8×10-8×104 U/ml of MuIFN / resulted in a dose dependent inhibition of cell proliferation measured by³H-thymidine incorporation and the MTT assay. The significant decrease of the CS (p < 0.008) but not the HA level, (measured densitometrically), was observed following 72 hours (hrs) incubation of G-26 cells with 8 × 10³ U/ml of MuIFN / (IFN treated cells: 0.03 ± 0.007 integrated optical density (IOD); control cells: 0.07 ± 0.01 IOD). The decreased CS production may be the underlying cause of IFN mediated inhibition of glioma cell proliferation.     

Glioblastoma Multiforme in an Asian Population: Evidence for a Distinct Genetic Pathway

In Singapore astrocytic tumours occur in only 25% of patients with primary brain tumours compared to 40–60% in other series. Glioblastoma multiforme arises either de novo as a primary glioblastomas associated with epidermal growth factor receptor (EGFR) and mdm2 over-expression or as a secondary glioblastomas, through malignant progression from low-grade astrocytomas, associated with p53 mutations and PDGFR- over-expression. Using immunohistochemical methods and DNA sequencing, we studied our population of glioblastomas for over-expression of EGFR, mdm2, p53, and PDGFR- as well directly for mutations of the p53 gene. While levels of over-expression of EGFR and mdm2 were consistent with levels expected for primary glioblastomas, levels of p53 and PDGFR- were consistent with levels documented for secondary glioblastomas. Notably 96% of the samples over-expressed p53 as detected with monoclonal antibody pAb 240. Of the 39 samples available for DNA sequencing 18% (7/39) had p53 mutations, including three mutations previously undocumented in glioblastomas. These results provide strong evidence that glioblastomas in Asian patients do not conform to currently accepted models of glioblastoma development, and that clinically defined glioblastomas in these patients show genetic changes consistent with both primary and 'secondary glioblastomas.     

Molecular Cloning of the Rat IL-13 Alpha 2 Receptor CDNA and Its Expression in Rat Tissues

The interleukin 13 alpha 2 receptor (IL-13R2) is highly expressed in human glioma cells. As a consequence this receptor has been proposed as a potential target for immunotherapeutic approaches for treating brain tumors. In developing animal models that may utilize the IL-13R2 receptor as an immunotherapeutic target, only the murine gene sequence has thus far been elucidated. The purpose of the present study, therefore, was to determine the gene sequence and tissue distribution of IL-13R2 in the rat. A search of the NCBI expressed sequence tag (EST) database with human and mouse IL-13R2 gene sequences identified a rat EST with high homology to the human and mouse IL-13R2 conserved region. Based on the sequence information, a 1917 bp rat IL-13R2 cDNA was cloned using the 5 and 3 RACE PCR technique. The cloned rat IL-13R2 cDNA contains a full-length 1158 bp open reading frame. The deduced protein is 91.2% and 54.2% homologous to mouse and human IL-13R2, respectively, at the amino acid level. Analysis shows that the rat IL-13R2 is structurally conserved and similar to human and mouse. It has a very short cytoplasmic domain, an extracellular domain containing an N-terminal fibronectin type III domain, four putative N-glycosylation sites, and a growth factor and cytokine receptor family motif WSEWS. Using RT-PCR techniques, the mRNA of rat IL-13R2 was detected in rat brain, spleen, liver, thymus, stomach, testis, and three rat glioblastoma cell lines C6, A15A5 and 9L. The cloning of rat IL-13R2 may be helpful to establish a rat model for IL-13R2 related glioma therapies.     

Comparative in vitro cytotoxic effects of taxol and its major human metabolite 6α-hydroxytaxol

Taxol is metabolized by the liver microsomal cytochrome P450 enzyme system into its principal metabolite 6-hydroxytaxol (6HT). In the present in vitro studies 6HT was compared to taxol with respect to its effects on tubulin depolymerization, mitotic arrest, clonogenic survival and apoptosis in HL-60 cells. 6HT was generated by incubating taxol with human liver microsomes in a NADPH-generating system. HL-60 cells were incubated for 24 h with either taxol or 6HT, washed and placed in drug-free suspension or cultured for colony growth in agarose. For the suspension and colony culture growth of the cells, the IC₅₀ concentrations of 6HT were 500±46 and 350±37 nM, while those of taxol were 3.2±0.2 and 2.8±0.5 nM, respectively. Immediately after a 24-h exposure of HL-60 cells to 50 nM taxol, electrophoresis of genomic DNA from HL-60 cells revealed an internucleosomal DNA fragmentation ladder. In addition, 39% of the cells were arrested in mitosis and 16% showed the morphologic features of apoptosis. In contrast, an identical treatment with 6HT resulted in the mitotic arrest of only 2.8% of the cells, with 4.0% displaying apoptosis (P<0.01); internucleosomal DNA fragmentation was not observed. 6HT was also significantly less effective than taxol in inhibiting the temperature-induced depolymerization of microtubules in a cell-free system. However, at equipotent concentrations, the effect of 6HT on tubulin depolymerization, mitotic arrest or apoptosis was similar to that of taxol. In addition, at concentrations of taxol or 6HT at or below their IC₅₀, there was little tubulin depolymerization, mitotic arrest or apoptosis. The results presented here show that the biotransformation of taxol to 6HT substantially detoxifies taxol.Abbreviations 6HT 6-hydroxytaxol - DMSO dimethyl sulfoxide - NADPH nicotinamide adenine dinucleotide phosphate - HPLC high-performance liquid chromatography - FBS fetal bovine serum - PBS phosphate-buffered saline - EDTA ethylenediaminetetraacetic acid - HEPES N-[2-hydroxyethyl]piperazine-N-2-ethanesulfonic acid - TAE tris-acetate EDTA - GTP guanosine triphosphate - MES 2-(N-morpholino) ethanesulfonic acid - EGTA ethylene glycol-bis(-aminoethylether)N,N,N,N-tetraacetic acid     

Genetic polymorphisms of platelet adhesive molecules: association with breast cancer risk and clinical presentation

The main platelet adhesive receptors integrin 21, integrin IIb3 and glycoprotein (GP) Ib are also expressed in breast carcinoma cells. They play a key role in tumor cell-induced platelet aggregation and in adhesive interactions necessary for tumoral invasion and metastasis. Several polymorphisms affecting these molecules, two in integrin 2 (C807T and G1648A), one in integrin 3 (T1565C) and one in GP Ib (VNTR), influencing their levels, structure, and possibly their function, have been previously described and associated with cardiovascular diseases. In this study, we investigated the association of these polymorphisms with breast cancer risk or clinical presentation. We studied 101 patients with invasive breast cancer. The main prognostic variables were recorded, and genomic PCR analysis of these polymorphisms was performed. A group of 101 control subjects matched on age and sex was studied and compared with patients. No association was found between VNTR (GP Ib) polymorphism and breast cancer risk or presentation. Genotype and allele frequencies of C807T and G1648A polymorphisms of integrin 2 were not statistically different in breast cancer patients and controls, although we found an association between the 1648G/G genotype and higher disease stages (III and IV) (p = 0.02). Breast cancer risk was higher in carriers of 3 integrin T/T genotype (OR = 2.08, 95% CI = 1.04–4.16, p = 0.04). Furthermore, genotype 1565T/T was also associated with axillary nodal metastasis (p = 0.017) and with tumoral diameter greater than 2 cm (p = 0.02). Although confirmatory studies are needed, our results suggest that polymorphic genetic variation of integrins expressed in platelets and epithelial breast cells could modify the risk and the biological aggressiveness of breast carcinomas.     

Blood-brain barrier changes following intracerebral injection of human recombinant tumor necrosis factor-α in the rat

Summary Human recombinant tumor necrosis factor- (rTNF-) was administered to normal Fischer 344 rats by stereotaxic intracerebral (IC) injection. Animals receiving a single injection of either 6 x 10⁴ U rTNF- or an equivalent volume of excipient (vehicle) in their right parietal lobe. In order to demonstrate any effects rTNF- might have on the blood-brain barrier (BBB), two studies were conducted, one employing exogenous horseradish peroxidase (HRP, 44 kD) as a tracer of BBB permeability and the other using endogenous IgG (150 kD). Rats given rTNF- showed transitory BBB permeability to HRP by 24 hours post-injection; this BBB compromise was determined to be no longer than 60 hours. In the other study, IgG was seen to cross the BBB by 48 hours post-rTNF- injection. Alternatively, rats injected IC with excipient showed only limited BBB opening as a result of injection-induced trauma. We conclude that human rTNF-, injected IC into normal rats triggers a temporary breakdown in BBB integrity which begins sometimes between 12 and 24 hours post-injection, is large enough to permit macromolecules of at least 150 kD to pass, and resolves by 72 hours post-injection.