Article

Alterações moleculares no carcinoma da mama e ovário: expressão de MCT1 e MCT4 e ampliação do HER2

Authors:
To read the full-text of this research, you can request a copy directly from the author.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the author.

ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
Several laboratories have investigated monocarboxylate transport in a variety of cell types. The characterization of the cloned transporter isoforms in a suitable expression system is nevertheless still lacking. H+/monocarboxylate co-transport was therefore investigated in monocarboxylate transporter 1 (MCT1)expressing.Xenopus laevis oocytes by using pH-sensitive microelectrodes and [C-14]lactate. Superfusion with lactate resulted in intracellular acidification of MCT1-expressing oocytes, but not in non-injected control oocytes. The basic kinetic properties of lactate transport in MCT1-expressing oocytes were determined by analysing the rates of intracellular pH changes under different conditions. The results were in agreement with the known properties of the transporter, with respect to both the dependence on the lactate concentration and the external pH value. Besides lactate, MCT1 mediated the reversible transport of a wide variety of monocarboxylic acids including pyruvate, D,L-3hydroxybutyrate, acetoacetate, alpha-oxoisohexanoate and alpha-oxoisovalerate, but not of dicarboxylic and tricarboxylic acids. The inhibitor alpha-cyano-4-hydroxycinnamate bound strongly to the transporter without being translocated, but could be displaced by the addition of lactate. In addition to changes in the intracellular pH, lactate transport also induced deviations from the resting membrane potential.
Article
Full-text available
Four transmembrane tyrosine kinases constitute the ErbB receptor family: the epidermal growth factor (EGF) receptor, ErbB-2, ErbB-3, and ErbB-4. We have measured the endocytic capacities of all four members of the EGF receptor family, including ErbB-3 and ErbB-4, which have not been described previously. EGF-responsive chimeric receptors containing the EGF receptor extracellular domain and different ErbB cytoplasmic domains (EGFR/ErbB) have been employed. The capacity of these growth factor-receptor complexes to mediate I-125-EGF internalization, receptor down-regulation, receptor degradation, and receptor co-immunoprecipitation with AP-2 was assayed. In contrast to the EGF receptor, all EGFR/ErbB receptors show impaired ligand-induced rapid internalization, down-regulation, degradation, and AP-2 association. Also, we have analyzed the heregulin-responsive wild-type ErbB-4 receptor, which does not mediate the rapid internalization of I-125-heregulin, demonstrates no heregulin-regulated receptor degradation, and fails to form association complexes with AP-2. Despite the substantial differences in ligand-induced receptor trafficking between the EGF and ErbB-4 receptors, EGF and heregulin have equivalent capacities to stimulate DNA synthesis in quiescent cells. These results show that the ligand-dependent down-regulation mechanism of the EGF receptor, surprisingly, is not a property of any other known ErbB receptor family member. Since endocytosis is thought to be an attenuation mechanism for growth factor-receptor complexes, these data imply that substantial differences in attenuation mechanisms exist within one family of structurally related receptors.
Article
Full-text available
ERBB2 (HER-2/neu) amplification and/or overexpression are associated with poor prognosis in node-positive breast carcinoma. Its prognostic value in node-negative cases and its predictive value for response to chemotherapy remain controversial. This may be related to the use of molecular methods, which are sensitive to dilution of tumor material by normal cells, or the use of nonstandardized immunohistochemistry (IHC) procedures, for the determination of the ERBB2 gene status. In addition, new therapeutic approaches that target the cells overexpressing ERBB2 are under development. These perspectives necessitate a reliable evaluation of the status of ERBB2 in individual tumors before the application of specific therapeutic strategies. Fluorescent in situ hybridization (FISH) and IHC allow the evaluation of the ERBB2 status specifically in tumor cells on archival material. We have analyzed a series of 100 invasive ductal breast carcinomas without lymph node invasion both by IHC, using the CB11 monodonal antibody and a sensitive Auidin Biotin Complex (ABC) immunodetection system, and by FISH, using the Oncor Inform HER-2/neu (ERBB2) gene amplification detection system as reference technique. Complete concordance between the results of FISH and IHC was seen in 98% of the cases. ERBB2 amplification (more than four signals per nucleus) was observed in 12 of the 100 cases, and all but one showed an overexpression of the protein (membrane staining) by IHC. Conversely, ERBB2 expression was present in one case without gene amplification. In conclusion, ERBB2 overexpression detected by IHC is highly correlated to gene amplification detected by FISH. Thus, under standardized conditions, IHC is a reliable and economical test to assess the ERBB2 status in tumors. The use of FISH could be limited to the verification of the status of tumors displaying a weak membrane immunostaining.
Article
Full-text available
The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1-4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1-4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including gamma-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and L: -lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.
Article
Full-text available
A novel potential cell surface receptor of the tyrosine kinase gene family has been identified and characterized by molecular cloning. Its primary sequence is very similar to that of the human epidermal growth factor receptor and the v-erbB oncogene product; the chromosomal location of the gene for this protein is coincident with the neu oncogene, which suggests that the two genes may be identical.
Article
Full-text available
Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin) is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells.
Article
Full-text available
The arbitrarily primed polymerase chain reaction (AP-PCR) [Welsh, J. & McClelland, M. (1990) Nucleic Acids Res. 18, 7213-7218] has been used to detect somatic genetic alterations in tumors of the colon and rectum. DNA fingerprints generated by single arbitrary primers were compared between normal and tumor tissue of the same individuals. AP-PCR bands showing decreased and increased intensities in tumor tissue DNA, relative to normal, have been cloned after reamplification with the same arbitrary primer. Standard restriction fragment length polymorphism and Southern blot analyses show that these DNA sequences have undergone allelic losses and gains, respectively, in the tumor cell genome. The deleted sequences have been assigned to the short arm of chromosome 17 by PCR of somatic hamster/human cell hybrids and linkage analysis. These results show the ability of the AP-PCR to detect and isolate, in a single step, DNA sequences representing two of the genetic alterations that underlie the aneuploidy of cancer cells: losses of heterozygosity and chromosomal gains. Altogether, they also show the quantitative nature of the amplification levels obtained in vitro by AP-PCR, which thus provides the basis for an alternative molecular approach to cancer cytogenetics.
Article
Full-text available
Article
Full-text available
Mathematical models developed from population ecology are applied to tumor-host interactions and demonstrate the importance of increased efficiency in substrate absorption as a mechanism enabling tumor cells to (a) proliferate despite inefficient energy production and (b) compete successfully for resources with the numerically superior host cells. As with many biological invasions observed in nature, success of the invaders can be enhanced by disruption of the local ecology if the disruption results in decreased viability of the native populations reducing their ability to inhibit tumor cell growth either directly through an immunological response or indirectly by competition with the tumor cells for available resources. Following successful invasion and displacement of normal cells from a volume of tissue, tumor cells achieve new equilibrium states with the environment based on their efficiency of consumption and the vascularity of the tissue. Tumor therapy may be enhanced by reducing available resources below a level which will support growth of the tumor cells or above a threshold which will allow repopulation by normal cells.
Article
Full-text available
Carcinoma of the breast and ovary account for one-third of all cancers occurring in women and together are responsible for approximately one-quarter of cancer-related deaths in females. The HER-2/neu proto-oncogene is amplified in 25 to 30 percent of human primary breast cancers and this alteration is associated with disease behavior. In this report, several similarities were found in the biology of HER-2/neu in breast and ovarian cancer, including a similar incidence of amplification, a direct correlation between amplification and over-expression, evidence of tumors in which overexpression occurs without amplification, and the association between gene alteration and clinical outcome. A comprehensive study of the gene and its products (RNA and protein) was simultaneously performed on a large number of both tumor types. This analysis identified several potential shortcomings of the various methods used to evaluate HER-2/neu in these diseases (Southern, Northern, and Western blots, and immunohistochemistry) and provided information regarding considerations that should be addressed when studying a gene or gene product in human tissue. The data presented further support the concept that the HER-2/neu gene may be involved in the pathogenesis of some human cancers.
Article
The uptake of laF-Deoxyglucose (FDG) was studied in vivo in relation to the proliferation rate of human head and neck tumors. Forty-two patients with histologically proven squa-mous-cell carcinoma of the head and neck and four patients with metastases of head and neck tumors were examined with PET and FDG prior to surgery. In 35 of these patients, a flow cytometric analysis of the DNA content and the proliferation rate was done using one-dimensional flow cytometry (DAPI staining). In 17 cases, perfusion studies with 1sO-labeled water were performed. Twenty-seven specimens were evaluable by flow cytometry. The analysis of the distribution of the FDG uptake revealed two groups, showing a high and a lower uptake pattern. In both groups the FDG uptake and the proliferation rate were correlated with an r-value of 0.64 and 0.8 respectively. However, the slope of the regression function was flat. No correlation was found between the perfusion and the proliferation rate. It is suggested that these differences in uptake in histologically identical tumor populations may correspond to differences at the molecular level, e.g., differences in the amount of the glucose carrier, perhaps caused by oncogenic transformation.
Article
PURPOSE: The objective of our study was to compare the methods used in the literature to analyze HER-2/neu status on archival breast cancer tissue. Therefore, a series of antibodies was evaluated to assess their immunohistochemical (IHC) sensitivity in correlation to gene amplification determined by fluorescence in situ hybridization (FISH). MATERIALS AND METHODS: HER-2/neu overexpression was studied on paraffin sections of 85 invasive breast cancers using a panel of five monoclonal (9G6, 3B5, CB11, TAB250, GSF-HER2) and two polyclonal antibodies (A8010, A0485) in addition to the HercepTest (DAKO, Glostrup, Denmark). HER-2/neu gene amplification was determined by FISH using a dual-color probe (PathVysion; Vysis, Stuttgart-Fasanenhof, Germany). RESULTS: HER-2/neu overexpression was demonstrated in 26% (9G6, TAB250, GSF-HER2), 27% (3B5, CB11), 33% (A8010) and 42% (A0485, HercepTest) of the tumors. FISH on paraffin sections identified gene amplification in 28% of the tumors. Strongly positive IHC results (3+) were always associated with gene amplification. Among the 16 tumors presented with weakly positive IHC results (2+) using the HercepTest, 12 (75%) lacked gene amplification. CONCLUSION: The comparison of IHC and FISH demonstrated an excellent correlation of high-level HER-2/neu overexpression (3+) with gene amplification; ie, FISH does not provide further information in these tumors. However, weakly positive IHC results (2+) obtained with the HercepTest share only a minor association with gene amplification.
Article
PURPOSE Decisions concerning the use of hormone therapy to treat metastatic breast cancer are made on the basis of the presence of estrogen receptor (ER). Despite the presence of ER, half of patients will not respond to hormone treatment. The purpose of this study was to determine the effect of overexpression of HER-2/neu on the response to hormone therapy. PATIENTS AND METHODS Sera from 300 metastatic breast cancer patients with ER-positive (ER+), ER status unknown, or ER-/progesterone receptor-positive (PR+) randomized to receive second-line hormone therapy with either megestrol acetate or fadrozole were evaluated. An enzyme immunoassay (EIA) specific for the extracellular domain of the c-erbB-2 (HER-2/neu) oncogene product was used to detect serum levels. RESULTS Fifty-eight patients (19.3%) had elevated serum c-erbB-2 protein levels, using a selected cut-point of 30 U/mL. The response rate (complete responses [CRs] plus partial responses [PRs] plus stable disease [S]) to endocrine therapy was 40.9% in 242 patients with low serum c-erbB-2 levels and only 20.7% in 58 patients with elevated serum c-erbB-2 levels (P = .004). The median duration of treatment response was longer in the group with low serum c-erbB-2 levels (15.5 months) compared with the group with elevated serum c-erbB-2 levels (11.6 months). Survival was also significantly shorter in patients with elevated serum c-erbB-2 levels (P < .0001). CONCLUSION Patients with ER+/c-erbB-2+ metastatic breast cancer are less likely to respond to hormone treatment than ER+/c-erbB-2- patients. Their survival duration is also shorter.
Article
Immunohistochemistry (IHC) is used commonly for evaluating HER-2/neu protein expression in breast cancer. Given the potential clinical importance of HER-2/neu status in patient management, interlaboratory variability in HER-2/neu IHC results is a matter of legitimate concern. We compared the results from 2 laboratories for HER-2/neu determined by IHC on paraffin sections of the same 100 consecutive invasive breast cancers. Both laboratories used the same primary antibody; however, different methods for heat-induced epitope retrieval (microwave or steam) and immunostaining (automated equipment from different manufacturers) and different scoring systems (positive-negative and 0-4+) were used. Slides were read in a blinded fashion and the results from the 2 laboratories were compared. Of the 93 cases evaluable in both laboratories, 24% were scored as HER-2/neu-positive at 1 laboratory, and 23% were scored as positive at the other. Complete concordance in categorization of HER-2/neu status between the 2 laboratories was achieved in 90 of 93 cases. Excellent interlaboratory agreement for HER-2/new IHC was attained suing the same primary antibody to HER-2/new, even without standardization of assay method or scoring criteria. However, standardization of these parameters remains an important objective to optimize interlaboratory agreement.
Book
Pediatric Urology is an up-to-date, clinical reference that provides detailed descriptions of the best approaches for the functional, biological, and morphological aspects of the urinary tract before and after birth. John G. Gearhart, Richard C. Rink, and Pierre D. E. Mouriquand cover all areas of the field, including pediatric surgery, radiology, nephrology, endocrinology, biochemistry, and obstetrics. Access the latest research through new chapters on tissue engineering, acute scrotum, and more. The appealing new full-color design, streamlined approach, and online access at expertconsult.com make this an invaluable resource to pediatric urologists, pediatric surgeons, residents and fellows worldwide. Provides detailed descriptions of the best approaches for the functional, biological, and morphological aspects of the urinary tract before and after birth. Features the fully searchable text online at expertconsult.com for convenient reference. Includes new chapters on tissue engineering, acute scrotum and disorders of the penis, and perinatal urological emergencies to cover the most up-to-date research in the field. Presents comprehensive coverage in a short, readable, and succinct format so that the material is easy to locate and disseminate. Provides cutting edge coverage from editors at the forefront of the specialty so you know the best available approaches. Eases reference and visual understanding through an all-new full-color design.
Book
Fish - including cyclostomes, elasmobranchs, and teleosts - display a wide variety of ways of coping with the stringent conditions they encounter in the aquatic environment. This diversity is especially evident in the female genital systems where some fish spawn profligate numbers of small eggs while others develop a few yolky eggs that may receive parental care. Parental care is carried to extremes in viviparity where eggs develop within the ovary or oviducts of the mother. This volume describes the myriad ways in which fish have approached problems of reproduction - it is an amply illustrated comparative study of the microscopic structure of the female genital systems of fish. The timing of its appearance is auspicious in that it coincides with the decline of the golden age of descriptive morphology. It is a compilation of thousands of micrographs - mostly electron micrographs - from classic works in the field and should prove valuable to investigators studying fish in areas such as ecology, physiology, and reproductive biology who may view histology as essential in their work but have little background in this area. It includes chapters on the origin of genital systems, the structure of ovarian follicles, mechanisms of ovulation, the cortical reaction, oviducts, oviparity, and amazing examples of viviparity.
Article
Monocarboxylates such as lactate and pyruvate play a central role in cellular metabolism and metabolic communication between tissues. Essential to these roles is their rapid transport across the plasma membrane, which is catalysed by a recently identified family of proton-linked monocarboxylate transporters (MCTs). Nine MCT-related sequences have so far been identified in mammals, each having a different tissue distribution, whereas six related proteins can be recognized in Caenorhabditis elegans and 4 in Saccharomyces cerevisiae. Direct demonstration of proton-linked lactate and pyruvate transport has been demonstrated for mammalian MCT1-MCT4, but only for MCT1 and MCT2 have detailed analyses of substrate and inhibitor kinetics been described following heterologous expression in Xenopus oocytes. MCT1 is ubiquitously expressed, but is especially prominent in heart and red muscle, where it is up-regulated in response to increased work, suggesting a special role in lactic acid oxidation. By contrast, MCT4 is most evident in white muscle and other cells with a high glycolytic rate, such as tumour cells and white blood cells, suggesting it is expressed where lactic acid efflux predominates. MCT2 has a ten-fold higher affinity for substrates than MCT1 and MCT4 and is found in cells where rapid uptake at low substrate concentrations may be required, including the proximal kidney tubules, neurons and sperm tails. MCT3 is uniquely expressed in the retinal pigment epithelium. The mechanisms involved in regulating the expression of different MCT isoforms remain to be established. However, there is evidence for alternative splicing of the 5'- and 3'-untranslated regions and the use of alternative promoters for some isoforms. In addition, MCT1 and MCT4 have been shown to interact specifically with OX-47 (CD147), a member of the immunoglobulin superfamily with a single transmembrane helix. This interaction appears to assist MCT expression at the cell surface. There is still much work to be done to characterize the properties of the different isoforms and their regulation, which may have wide-ranging implications for health and disease. In the future it will be interesting to explore the linkage of genetic diseases to particular MCTs through their chromosomal location.
Article
Associations of reproductive factors with ovarian cancer may differ by histologic type. Data from a multiethnic, population-based, case-control study, conducted in Hawaii and Los Angeles, California, between 1993 and 1999, were used to assess this hypothesis. A structured questionnaire was administered to 558 histologically confirmed epithelial ovarian cancer cases and 607 population controls. Factors suppressing ovulation, including pregnancy and oral contraceptive use, were inversely associated with the risk of all histologic types. Nonmucinous but not mucinous tumors were significantly associated with menstruation years (odds ratio = 1.5 for the highest vs. the lowest quartile) and lifetime ovulatory cycles (odds ratio = 2.8 for the highest vs. the lowest quartile). Duration of breastfeeding (odds ratio = 0.4 for the highest vs. the lowest quartile) was significantly and inversely related to nonmucinous tumors but not to mucinous tumors. Among all tumor types, endometrioid tumors were the most strongly related to pregnancy and tubal ligation, while clear cell tumors were the only type that was associated with noncontraceptive hormone use. The risk factors were similar for borderline and invasive tumors, except for age at diagnosis. Mucinous tumors, both borderline and invasive, were more common in Asian women than in Caucasian and other women. Our data suggest that histologic types of epithelial ovarian cancer are etiologically distinct. breast feeding; case-control studies; contraceptives, oral; histology; ovarian neoplasms; ovulation; parity; pregnancy
Book
DeVita, Hellman, and Rosenberg's Cancer: Principles & Practice of Oncology, 10th edition has garnered universal acclaim as the world's definitive, standard-setting oncology reference. More than 400 respected luminaries explore today's most effective strategies for managing every type of cancer by stage of presentation - discussing the role of all appropriate therapeutic modalities as well as combined-modality treatments. This multidisciplinary approach will help your cancer team collaboratively face the toughest clinical challenges and provide the best possible care for every cancer patient. Access the complete contents online or on your mobile device, with quarterly updates reflecting late-breaking developments in cancer care on LWW Health Library. Take full advantage of the latest advances with brand-new chapters on Hallmarks of Cancer, Molecular Methods in Cancer, Oncogenic Viruses, Cancer Screening, and new sections on Genetic testing and counseling for cancer, plus comprehensive updates throughout - including coverage of the newest biologic therapies. Make optimal, well-coordinated use of all appropriate therapies with balanced, multidisciplinary advice from a surgeon, a medical oncologist, and a radiation oncologist in each major treatment chapter. Review the latest molecular biology knowledge for each type of cancer and its implications for improved management. Make the best decisions on cancer screening and prevention, palliative care, supportive oncology, and quality-of-life issues. Access and receive quarterly content updates for Cancer: Principles and Practice, 10th Edition on the NEW and ENHANCED LWW Health Library while on the go. Additionally, for the first 6 months, Cancer: Principles and Practice of Oncology, 10th Edition purchasers will get access to the entire Oncology Collection on LWW Health Library - an incredible value at no additional charge!. © 2011 by Wolters Kluwer Health / Lippincott Williams & Wilkins. All rights reserved.
Article
The differences between the mammary glands of rhesus monkeys and human breasts are mostly in their gross structure: otherwise, their glandular mechanisms and innervation are nearly identical.
Article
Many genes and signalling pathways controlling cell proliferation, death and differentiation, as well as genomic integrity, are involved in cancer development. New techniques, such as serial analysis of gene expression and cDNA microarrays, have enabled measurement of the expression of thousands of genes in a single experiment, revealing many new, potentially important cancer genes. These genome screening tools can comprehensively survey one tumor at a time; however, analysis of hundreds of specimens from patients in different stages of disease is needed to establish the diagnostic, prognostic and therapeutic importance of each of the emerging cancer gene candidates. Here we have developed an array-based high-throughput technique that facilitates gene expression and copy number surveys of very large numbers of tumors. As many as 1000 cylindrical tissue biopsies from individual tumors can be distributed in a single tumor tissue microarray. Sections of the microarray provide targets for parallel in situ detection of DNA, RNA and protein targets in each specimen on the array, and consecutive sections allow the rapid analysis of hundreds of molecular markers in the same set of specimens. Our detection of six gene amplifications as well as p53 and estrogen receptor expression in breast cancer demonstrates the power of this technique for defining new subgroups of tumors.
Article
Improvements in a method for the specific microscopic localization of antigen in tissue cells are described. This method employs antibody labelled with fluorescein isocyanate as a histochemical stain, the specific antigen-antibody precipitate being made visible under the fluorescence microscope. Two isomeric series derived from nitrofluorescein are described.
Article
A hybrid cellular automaton model is described and used to simulate early tumor growth and examine the roles of host tissue vascular density and tumor metabolism in the ability of a small number of monoclonal transformed cells to develop into an invasive tumor. The model incorporates normal cells, tumor cells, necrotic or empty space, and a random network of native microvessels as components of a cellular automaton state vector. Diffusion of glucose and H+ions (the latter largely resulting from the tumor's excessive reliance on anaerobic metabolism) to and from the microvessels, and their utilization or production by cells, is modeled through the solution of differential equations. In this way, the cells and microvessels affect the extracellular concentrations of glucose and H+which, in turn, affect the evolution of the automaton. Simulations of the model demonstrate that: (i) high tumor H+ion production is favorable for tumor growth and invasion; however for every H+ion production rate, there exists a range of optimal microvessel densities (leading to a local pH favorable to tumor but not to normal cells) for which growth and invasion is most effective, (ii) at vascular densities below this range, both tumor and normal cells die due to excessively low pH, (iii) for vascular densities above the optimal range the microvessel network is highly efficient at removing acid and therefore the tumor cells lose their advantage over normal cells gained by high local H+concentration. While significant spatial gradients of glucose formed, no regions of detrimentally poor glucose perfusion (for either cell type) were observed, regardless of microvessel density. Depending on metabolic phenotype, a variety of tumor morphologies similar to those clinically observed were realized in the simulations. Lastly, a sharp transition (analogous to that of the adenoma–carcinoma sequence) between states of initial tumor confinement and efficient invasiveness was observed when H+production reached a critical value.
Article
1Monocarboxylate transporter (MCT) 4 is the major monocarboxylate transporter isoform present in white skeletal muscle and is responsible for the efflux of lactic acid produced by glycolysis. Here we report the characterisation of MCT4 expressed in Xenopus oocytes.2The protein was correctly targeted to the plasma membrane and rates of substrate transport were determined from the rate of intracellular acidification monitored with the pH-sensitive dye 2′,7′-bis-(carboxyethyl)-5(6)-carboxyfluorescein (BCECF).3In order to validate the technique, the kinetics of monocarboxylate transport were measured in oocytes expressing MCT1. Km values determined for L-lactate, D-lactate and pyruvate of 4.4, > 60 and 2.1 mM, respectively, were similar to those determined previously in tumour cells.4Comparison of the time course of [14C]lactate accumulation with the rate of intracellular acidification monitored with BCECF suggests that the latter reflects pH changes close to the plasma membrane associated with transport, whilst the former may include diffusion-limited movement of lactate into the bulk cytosol.5Km values of MCT4 for these substrates were found to be 28, 519 and 153 mM, respectively, and for a range of other monocarboxylates values were at least an order of magnitude higher than for MCT1. Vmax values appeared to be similar for all substrates.6K0.5 values of MCT4 (determined at 30 mM L-lactate) for inhibition by α-cyano-4-hydroxycinnamate (991 μM), phloretin (41 μM), 5-nitro-2-(3-phenylpropylamino)benzoate (240 μM), p-chloromercuribenzene sulphonate (21 μM) and 3-isobutyl-1-methylxanthine (970 μM, partial inhibition) were also substantially higher than for MCT1. No inhibition of MCT4 by 2 mM 4,4′-diisothiocyanostilbene-2,2′-disulphonate was observed.7The properties of MCT4 are consistent with published data on giant sarcolemmal vesicles in which MCT4 is the dominant MCT isoform, and are appropriate for the proposed role of MCT4 in mediating the efflux from the cell of glycolytically derived lactic acid but not pyruvate.
Article
• Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription-polymerase chain reaction (RT-PCR). The sequence of the RT-PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT-PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well-characterized human and pig colonic lumenal membrane vesicles (LMV). • Properties of the colonic lumenal membranel-lactate transporter were studied by the uptake of L-[U-14C]lactate into human and pig colonic LMV.l-lactate uptake was stimulated in the presence of an outward-directed anion gradient at an extravesicular pH of 5.5. Transport ofl-lactate into anion-loaded colonic LMV appeared to be via a proton-activated, anion exchange mechanism. • l-lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl− and SO42−. The uptake ofl-lactate was inhibited by phloretin, mercurials and α-cyano-4-hydroxycinnamic acid (4-CHC), but not by the stilbene anion exchange inhibitors, 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS) and 4-acetamido-4′-isothiocyanostilbene-2,2′-disulphonic acid (SITS). • The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport ofl-lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.
Article
Recent years have witnessed tremendous growth in the epidermal growth factor (EGF) family of peptide growth factors and the ErbB family of tyrosine kinases, the receptors for these factors. Accompanying this growth has been an increased appreciation for the roles these molecules play in tumorigenesis and in regulating cell proliferation and differentiation during development. Consequently, a significant question has been how diverse biological responses are specified by these hormones and receptors. Here we discuss several characteristics of hormone-receptor interactions and receptor coupling that contribute to specificity: 1) a single EGF family hormone can bind multiple receptors; 2) a single ErbB family receptor can bind multiple hormones; 3) there are three distinct functional groups of EGF family hormones; 4) EGF family hormones can activate receptors in trans, and this heterodimerization diversifies biological responses; 5) ErbB3 requires a receptor partner for signaling; and 6) ErbB family receptors differentially couple to signaling pathways and biological responses. BioEssays 20:41–48, 1998. © 1998 John Wiley & Sons, Inc.
Article
Monocarboxylate transporter 2 (MCT2) expression is up-regulated by noradrenaline (NA) in cultured cortical neurons via a putative but undetermined translational mechanism. Western blot analysis showed that p44/p42 mitogen-activated protein kinase (MAPK) was rapidly and strongly phosphorylated by NA treatment. NA also rapidly induced serine/threonine protein kinase from AKT virus (Akt) phosphorylation but to a lesser extent than p44/p42 MAPK. However, Akt activation persisted over a longer period. Similarly, NA induced a rapid and persistent phosphorylation of mammalian target of rapamycin (mTOR), a kinase implicated in the regulation of translation in the central nervous system. Consistent with activation of the mTOR/S6 kinase pathway, phosphorylation of the ribosomal S6 protein, a component of the translation machinery, could be observed upon treatment with NA. In parallel, it was found that the NA-induced increase in MCT2 protein was almost completely blocked by LY294002 (phosphoinositide 3-kinase inhibitor) as well as by rapamycin (mTOR inhibitor), while mitogen-activated protein kinase kinase and p38 MAPK inhibitors had much smaller effects. Taken together, these data reveal that NA induces an increase in neuronal MCT2 protein expression by a mechanism involving stimulation of phosphoinositide 3-kinase/Akt and translational activation via the mTOR/S6 kinase pathway. Moreover, considering the role of NA in synaptic plasticity, alterations in MCT2 expression as described in this study might represent an adaptation to face energy demands associated with enhanced synaptic transmission.
Article
Seventeen patients with intracranial meningiomas were studied with positron emission tomography and fluorine-18-2-fluorodeoxyglucose (PET-FDG) to assess the glucose utilization of these tumors. Four meningiomas followed for 3-5 years after PET-FDG and surgery showed no evidence of recurrence. These tumors had significantly lower glucose utilization rates (1.9 mg/dl/min +/- 1.0) than 11 recurrent or regrowing meningiomas (4.5 mg/dl/min +/- 1.96) (P less than .01). The glucose metabolic rates of meningiomas correlated with tumor growth, as estimated from changes in tumor size on repeated computed tomographic scans. Histopathologically, a syncytial (atypical) meningioma had the highest glucose utilization rate, followed by a papillary meningioma and an angioblastic meningioma. Individual transitional and syncytial (typical) meningiomas showed marked differences in glucose metabolism despite similar microscopic appearance. Glucose utilization rate appears to be at least as reliable as histologic classification and other proposed criteria for predicting the behavior and recurrence of intracranial meningiomas.
Article
Inactivation of the von Hippel-Lindau (VHL) tumour-suppressor protein (pVHL) is associated with the von Hippel-Lindau cancer syndrome and the majority of kidney cancers. New evidence suggests that pVHL has properties of an F-box protein that targets the alpha-subunits of hypoxia-inducible factor (HIF)-1 for oxygen-dependent ubiquitination.
Article
The induction of hypoxia-inducible factor 1 (HIF-1) activity, either as a result of intratumoral hypoxia or loss-of-function mutations in the VHL gene, leads to a dramatic reprogramming of cancer cell metabolism involving increased glucose transport into the cell, increased conversion of glucose to pyruvate, and a concomitant decrease in mitochondrial metabolism and mitochondrial mass. Blocking these adaptive metabolic responses to hypoxia leads to cell death due to toxic levels of reactive oxygen species. Targeting HIF-1 or metabolic enzymes encoded by HIF-1 target genes may represent a novel therapeutic approach to cancer.
Article
Amplification of the HER-2 (c-erbB-2) gene and overexpression of the p185HER-2 gene product is found in approximately one-third of primary human breast and ovarian cancers and is associated with a poor clinical outcome of early relapse and death. The HER-2 gene encodes a cell-surface growth factor receptor with intrinsic tyrosine kinase activity. Wild-type human HER-2 has been shown to act as a potent oncogene when over-expressed in mouse fibroblasts. Recent data suggest that the mechanism by which HER-2 mediates transformation requires the interaction of the epidermal growth factor (EGF) receptor. To test whether overexpression of normal human HER-2 can transform cells independently of the EGF receptor, we have introduced multiple copies of HER-2 into the EGF receptor-negative cell line, NR6, and have performed assays for both transformation and tumorigenicity. Engineered NR6 cells that overexpress the HER-2 gene product display a highly transformed and tumorigenic phenotype as compared with control cells. Additionally, a monoclonal antibody to the extracellular domain of the HER-2 receptor is able to inhibit the proliferation of the overexpressing cells in vitro as well as tumor growth in vivo. This study provides clear evidence that HER-2-mediated transformation can be achieved independently of the EGF receptor.
Article
Overexpression and amplification of the neu (c-erbB2, ERBB2) protooncogene have been implicated in the development of aggressive human breast cancer. To directly assess the effect of mammary gland-specific expression of the neu protooncogene, transgenic mice carrying unactivated neu under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were established. By contrast to the rapid tumor progression observed in several transgenic strains carrying the activated neu transgene, expression of unactivated neu in the mammary epithelium resulted in the development of focal mammary tumors after long latency. The majority of the mammary tumors analyzed expressed elevated levels of neu-encoded mRNA and protein. Overexpression of neu in the mammary tumors was also associated with elevated neu intrinsic tyrosine kinase activity and the de novo tyrosine phosphorylation of several cellular proteins. Interestingly, many of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. These observations suggest that overexpression of the unactivated neu protein can induce metastatic disease after long latency.
Article
Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.
Article
The HER2 gene encodes a cell-surface glycoprotein with extensive homology to the epidermal growth factor receptor. Recently it was found to be amplified in about 30% of primary human breast malignancies. In experiments designed to assess the role of the HER2 gene in oncogenesis, we found that overexpression of unaltered HER2 coding sequences in NIH 3T3 cells resulted in cellular transformation and tumorigenesis.
Article
Barrett's esophagus is a condition in which metaplastic columnar epithelium replaces squamous esophageal epithelium as a consequence of chronic gastroesophageal reflux. Patients with this condition are at increased risk for the development of adenocarcinoma. To better understand the progression to adenocarcinoma in this disease, we studied abnormalities in DNA content of epithelial cells in Barrett's esophagus. Using flow cytometry, we examined the spatial distribution of abnormal nuclear DNA contents (aneuploidy) in the esophagi of 14 patients with Barrett's adenocarcinoma. Multiple (2 to 14) populations of aneuploid cells were seen in 12 of the 14 cases. Some early carcinomas appeared to be associated with a single aneuploid population of cells. Surrounding dysplastic epithelium often contained multiple, different overlapping aneuploid populations. These data suggest that neoplastic progression in Barret's esophagus is associated with a process of genomic instability which leads to evolution of multiple aneuploid populations, with the ultimate development of a clone of cells capable of malignant invasion. Thus, detection of multiple aneuploid populations of cells in Barrett's esophagus may indicate a high risk of cancer. Barrett's esophagus provides a unique and readily accessible model for the study of neoplastic progression in human epithelial malignancy.
Article
New methods were used to identify the abnormality in a patient who showed evidence of neuromuscular dysfunction on extensive clinical examination. The methods revealed that the lactate content of the patient's skeletal muscle does not decline normally after exercise and that his red cells are defective in lactate transport. These results suggest that skeletal muscle and erythrocyte membranes share the same genetic lactate transporter (or a common subunit), which is deficient in this patient. This defect may be a common cause of elevated serum creatine kinase levels, as seen in the patient described here and of unexplained episodes of rhabdomyolysis and myoglobinuria.
Article
Seven years after the initial studies of the prognostic value of the proto-oncogene c-erbB-2 in breast cancer, its role is still being defined. The interpretation of studies on the use of this gene and its protein product in prognostic and predictive tests for breast cancer is complicated by multiple methodologies and the inherent difficulties in the studies. The work has moved beyond the stage at which small studies with short follow-up (useful for hypothesis generation) are of value, to the stage in which large studies with sufficient statistical power to find significant correlations are central. These larger studies do not lend support for the use of c-erbB-2 in the evaluation of axillary-node-negative patients, the group of breast cancer patients for whom refinement of prognostic estimates is now most important. There are, however, hints that c-erbB-2 may have value in predicting response to certain treatments, though the studies so far are too few, often too small and too conflicting to reliably confirm this.
Article
A serine/threonine kinase, named protein kinase B (PKB) for its sequence homology to both protein kinase A and C, has previously been isolated. PKB, which is identical to the kinase Rac, was later found to be the cellular homologue of the transforming v-Akt. Here we show that PKB is activated by stimuli such as insulin, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Activation of PKB was inhibited by the phosphatidylinositol-3-OH kinase (PI(3)K) inhibitor wortmannin and by coexpression of a dominant-negative mutant of PI(3)K. PDGF receptor mutants that lack detectable associated PI(3)K activity also fail to induce PKB activation, PKB kinase activity is correlated with phosphorylation of PKB on serine. Finally, we show that a constructed Gag-PKB fusion protein, homologous to the v-akt oncogene, displays significantly increased ligand-independent kinase activity. Furthermore, this activity is sufficient to activate the p70 S6-kinase (p70S6K). These results suggest a role for PKB in PI(3)K-mediated signal transduction.
Article
A c-erbB-2 expression vector was transfected into the estrogen receptor positive (ER+) MCF-7 human breast cancer cell line to determine if overexpression of this transmembrane tyrosine kinase could increase the malignant phenotype of this cell line. Loss of transfected c-erbB-2 expression was observed when cells were carried in medium containing estrogen. Homogeneous populations stably overexpressing levels of the 185 kDa c-erbB-2 observed in the SKBR-3 a breast cancer cell line which overexpresses c-erbB-2 as a result of gene amplification could be obtained by continually maintaining the transfected cell lines in estrogen-free conditions. Levels of constitutively activated c-erbB-2 varied among clonal isolates. Whereas some overexpressing lines did acquire the ability to form transient tumor nodules in ovariectomized nude mice without estrogen supplementation, as well as in mice that received the antiestrogen tamoxifen, one cell line that exhibited the highest levels of constitutively activated c-erbB-2 was able to form static tumors of a larger size under both conditions. This same cell line formed progressively growing tumors in estrogen-supplemented mice that were much larger than observed in mice injected with control cell lines, and also showed reduced sensitivity to antiestrogens in vitro, but it continued to have a low metastatic phenotype. These results suggest that signal transduction mediated by the c-erbB-2 tyrosine kinase can partially overcome the estrogen dependence of ER+breast cancer cells for growth and that c-erbB-2 overexpression confers a selective advantage to such cells in the absence of estrogen.