Bacterial toxins and heart function: heat-labile Escherichia coli enterotoxin B promotes changes in cardiac function with possible relevance for sudden cardiac death.

Bacterial toxins can cause cardiomyopathy, though it is not its most common cause. Some bacterial toxins can form pores in the membrane of cardiomyocytes, while others can bind to membrane receptors. Enterotoxigenic E. coli can secrete enterotoxins, including heat-resistant (ST) or labile (LT) enterotoxins. LT is an AB5-type toxin that can bind to specific cell receptors and disrupt essential host functions, causing several common conditions, such as certain diarrhea. The pentameric B subunit of LT, without A subunit (LTB), binds specifically to certain plasma membrane ganglioside receptors, found in lipid rafts of cardiomyocytes. Isolated guinea pig hearts and cardiomyocytes were exposed to different concentrations of purified LTB. In isolated hearts, mechanical and electrical alternans and an increment of heart rate variability, with an IC50 of ~0.2 µg/ml LTB, were observed. In isolated cardiomyocytes, LTB promoted significant decreases in the amplitude and the duration of action potentials. Na+ currents were inhibited whereas L-type Ca2+ currents were augmented at their peak and their fast inactivation was promoted. Delayed rectifier K+ currents decreased. Measurements of basal Ca2+ or Ca2+ release events in cells exposed to LTB suggest that LTB impairs Ca2+ homeostasis. Impaired calcium homeostasis is linked to sudden cardiac death. The results are consistent with the recent view that the B subunit is not merely a carrier of the A subunit, having a role explaining sudden cardiac death in children (SIDS) infected with enterotoxigenic E. coli, explaining several epidemiological findings that establish a strong relationship between SIDS and ETEC E. coli. Supplementary Information: The online version contains supplementary material available at 10.1007/s12551-023-01100-6.

Lead poisoning: acute exposure of the heart to lead ions promotes changes in cardiac function and Cav1.2 ion channels.

Lead ions (Pb2+) possess characteristics similar to Ca2+. Because of this and its redox capabilities, lead causes different toxic effects. The neurotoxic effects have been well documented; however, the toxic effects on cardiac tissues remain allusive. We utilized isolated guinea pig hearts and measured the effects of Pb2+ on their contractility and excitability. Acute exposure to extracellular Pb2+ had a negative inotropic effect and increased diastolic tension. The speed of contraction and relaxation were affected, though the effects were more dramatic on the speed of contraction. Excitability was also altered. Heart beat frequency increased and later diminished after lead ion exposure. Pro-arrhytmic events, such as early after-depolarization and a reduction of the action potential plateau, were also observed. In isolated cardiomyocytes and tsA 201 cells, extracellular lead blocked currents through Cav1.2 channels, diminished their activation, and enhanced their fast inactivation, negatively affecting their gating currents. Thus, Pb2+ was cardiotoxic and reduced cardiac contractility, making the heart prone to arrhythmias. This was due, in part, to Pb2+ effects on the Cav1.2 channels; however, other channels, transporters or pathways may also be involved. Acute cardiotoxic effects were observed at Pb2+ concentrations achievable during acute lead poisoning. The results suggest how Cav1.2 gating can be affected by divalent cations, such as Pb2, and also suggest a more thorough evaluation of heart function in individuals affected by lead poisoning.

Multiple co-infections (Mycoplasma, Chlamydia, human herpes virus-6) in blood of chronic fatigue syndrome patients: association with signs and symptoms.

Previously we and others found that a majority of chronic fatigue syndrome (CFS) patients showed evidence of systemic mycoplasmal infections, and their blood tested positive using a polymerase chain reaction assay for at least one of the four following Mycoplasma species: M. fermentans, M. hominis, M. pneumoniae or M. penetrans. Consistent with previous results, patients in the current study (n=200) showed a high prevalence (overall 52%) of mycoplasmal infections. Using forensic polymerase chain reaction we also examined whether these same patients showed evidence of infections with Chlamydia pneumoniae (overall 7.5% positive) and/or active human herpes virus-6 (HHV-6, overall 30.5% positive). Since the presence of one or more infections may predispose patients to other infections, we examined the prevalence of C. pneumoniae and HHV-6 active infections in mycoplasma-positive and -negative patients. Unexpectedly, we found that the incidence of C. pneumoniae or HHV-6 was similar in Mycoplasma-positive and -negative patients, and the converse was also found in active HHV-6-positive and -negative patients. Control subjects (n=100) had low rates of mycoplasmal (6%), active HHV-6 (9%) or chlamydial (1%) infections, and there were no co-infections in control subjects. Differences in bacterial and/or viral infections in CFS patients compared to control subjects were significant. Severity and incidence of patients' signs and symptoms were compared within the above groups. Although there was a tendency for patients with multiple infections to have more severe signs and symptoms (p<0.01), the only significant differences found were in the incidence and severity of certain signs and symptoms in patients with multiple co-infections of any type compared to the other groups (p<0.01). There was no correlation between the type of co-infection and severity of signs and symptoms. The results indicate that a large subset of CFS patients show evidence of bacterial and/or viral infection(s), and these infections may contribute to the severity of signs and symptoms found in these patients.

Tumor cell adhesion under hydrodynamic conditions of fluid flow.

Current evidence indicates that tumor cell adhesion to the microvasculature in host organs during formation of distant metastases is a complex process involving various types of cell adhesion molecules. Recent results have shown that stabilization of tumor cell adhesion to the microvascular vessel wall is a very important step for successful tumor cell migration and colonization of host organs. We are beginning to understand the influences of fluid flow and local shear forces on these adhesive interactions and cellular responses within the circulation. Mechanosensory molecules or molecular complexes can transform shear forces acting on circulating tumor cells into intracellular signals and modulate cell signaling pathways, gene expression and other cellular functions. Flowing tumor cells can interact with microvascular endothelial cells mediated mainly by selectins, but the strength of these bonds is relatively low and not sufficient for stable cell adhesions. Integrin-mediated tumor cell adhesion and changes in the binding affinity of these adhesion molecules appear to be required for stabilized tumor cell adhesion and subsequent cell migration into the host organ. Failure of the conformational affinity switch in integrins results in breaking of these bonds and recirculation or mechanical damage of the tumor cells. Various cell signaling molecules, such as focal adhesion kinase, pp60src or paxillin, and cytoskeletal components, such as actin or microtubules, appear to be required for tumor cell adhesion and its stabilization under hydrodynamic conditions of fluid flow.

The role of tumor cell adhesion as an important factor in formation of distant colorectal metastasis.

PURPOSE: The interactions of blood-borne colorectal carcinoma cells with vascular endothelium are important during hematogenous formation of distant metastases. To adhere to the vessel wall, circulating carcinoma cells that come into contact with the microvasculature must resist the attractive forces of the flow of plasma and other circulating cells that tend to detach them from the wall. METHODS: Hydrodynamic adhesion assays have been introduced to mimic the microcirculation and investigate cell adhesion under flow conditions. Different aspects of colorectal cancer cell adhesion during hematogenous formation of distant metastases are summarized and discussed in this review. RESULTS: Adhesion of colorectal carcinoma cells to endothelial cells and extracellular matrix is influenced by the presence of fluid flow. Shear forces alone are able to induce signal transduction events in these cells that result in cell activation and modification of adhesive behavior. CONCLUSIONS: Consideration of fluid dynamics of circulating colorectal cancer cell movement in the microcirculation leads to new knowledge of in vivo processes that are involved in tumor cell adhesion to the vessel wall in host organs. Shear forces have been found to influence adhesive properties of colorectal carcinoma cells to endothelial cells and underlying subendothelial extracellular matrix. Understanding the complex processes involved in tumor cell adhesion may result in the development of novel therapeutic strategies.

Tumor metastasis-associated human MTA1 gene: its deduced protein sequence, localization, and association with breast cancer cell proliferation using antisense phosphorothioate oligonucleotides.

Using differential cDNA library screening techniques based on metastatic and nonmetastatic rat mammary adenocarcinoma cell lines we previously cloned and sequenced the metastasis-associated gene mta1. Using homology to the rat MTA1 gene we cloned the human MTA1 gene and found it to be overexpressed in a variety of human cell lines. We found a close similarity between the human MTA1 and rat MTA1 genes, as shown by 88% and 96% identities of the nucleotide and predicted amino acid sequences, respectively. Both genes encode novel proteins that contain a proline-rich region (SH3 binding motif), a putative zinc finger motif, a leucine zipper motif, and five copies of the SPXX motif often found in gene regulatory proteins. Using Southern blot analysis, the MTA1 gene was found to be highly conserved among all species examined; and using Northern blot analysis, MTA1 transcripts were found in virtually all cell lines of human origin that were analyzed, including melanoma and breast, cervix and ovarian carcinoma cells and normal breast epithelial cells. However, the expression level of the MTA1 gene in a normal breast epithelial cell was approximately 50% of that found in rapidly growing breast adenocarcinoma cell lines and an atypical mammary cell line. Experimental inhibition of MTA1 protein expression using antisense phosphorothioate oligonucleotides resulted in growth inhibition of human MDA-MB-231 breast cancer cells with relatively high expression of the MTA1 gene. Furthermore, the MTA1 protein was localized in the nuclei of cells transfected using a mammalian expression vector containing the full-length MTA1 gene. The results suggest that the MTA1 protein may function in cellular signaling processes important in the progression and growth of cancer cells, possibly as a nuclear regulatory factor.

Time-dependent dephosphorylation through serine/threonine phosphatases is required for stable adhesion of highly and poorly metastatic HT-29 colon carcinoma cell lines to collagen.

Adhesion stabilization is a prerequisite for the long-term adhesion of circulating metastatic tumor cells, and tumor cells with different metastatic potential demonstrate distinct patterns of cell adhesion properties. An important event during formation of organ metastases is integrin-mediated extracellular matrix (ECM) binding that can initiate signal transduction events. Recently we reported that Ser/Thr kinases are involved in regulation of tumor cell adhesion. In the present study the influence of dephosphorylation by Ser/Thr protein phosphatases (PPases) on tumor cell adhesion was investigated. Pretreatment of poorly and highly metastatic human HT-29 colon carcinoma cells with the broad-range inhibitors sodium fluoride (NaF) and sodium pyrophosphate (PyroP) resulted in strong reduction in adhesion of HT-29 cells to various ECM components. Surprisingly, when specific Ser/Thr PPase inhibitors like tautomycin were used we found only a partial reduction in adhesion of highly metastatic HT-29LMM cells to collagen I but not to collagen IV. Other inhibitors did not inhibit adhesion, and poorly metastatic HT-29P were not affected by any specific Ser/Thr PPase inhibitors. Therefore, the effects of NaF on adhesion-mediated Tyr phosphorylation were investigated further. Pretreatment with this inhibitor led to a reduction in phosphorylation of focal adhesion kinase (FAK). In contrast, in cells grown adherent to tissue culture dishes, low concentrations of NaF increased FAK phosphorylation whereas high concentrations inhibited the amount of phosphorylated FAK. Although NaF inhibited adhesions it did not cause changes in cell morphology or detachment of cells from ECM. We hypothesize that dual-specific PPases may be involved in the regulation and establishment of new adhesive interactions in HT-29 cells, but they are not required for maintenance of stable adhesions to ECM.

Molecular analysis of a candidate metastasis-associated gene, MTA1: possible interaction with histone deacetylase 1.

We previously identified a novel rat candidate metastasis-associated gene, mta1, based on its differential expression in highly metastatic cells compared to nonmetastatic cells. Furthermore, we showed that overexpression of its human counterpart, MTA1, correlated with the invasiveness or lymph node metastasis of gastric, colorectal and esophageal carcinomas. The aim of this study was to analyze the domains of the MTA1 and investigate the function(s) of this protein. Structural analysis revealed that the MTA1 protein contained a GATA-like zinc-finger domain, a leucine zipper domain, a SANT domain similar to the DNA binding domain of myb-related proteins, a src homology 3-binding domain important in protein-protein interactions, two highly acidic regions characteristic of the acidic activation domains of many transcription factors, and nuclear localization signals. Immunofluorescence staining of COS-7 cells transfected with a myc-epitope-tagged MTA1 expression vector clearly showed nuclear localization of MTA1. Coimmunoprecipitation of myc-tagged MTA1 and FLAG-tagged histone deacetylase 1 (HDAC1), followed by western blot analysis using anti-myc and anti-FLAG monoclonal antibodies showed that MTA1 physically bound with HDAC1 in COS-7 cells. Together with the recent finding that the NURD (nucleosome remodeling and histone deacetylase activities) complex contains an MTA1-related gene product, named MTA2, MTA1 may be another component of this complex and be involved in the alteration of chromatin structure and transcription repression.

Cell surface molecules and their prognostic values in assessing colorectal carcinomas.

OBJECTIVE: Carcinomas of the colon and rectum are the third leading cause of cancer-related deaths. Although advances in the surgical treatment of primary colorectal cancers have lead to improvements in patient survival at early tumor stages, treatment of more progressive cancers has not resulted in dramatic improvements in patient survival. However, the selection of patient subgroups based on their prognosis and other characteristics could result in improved outcomes from adjuvant therapies in patients with Dukes B and C carcinomas. METHODS: The authors reviewed the available data on the value of cell surface molecules in assessing the prognosis of colorectal carcinomas, paying specific attention to the evaluation of statistical analysis and multivariate procedures. RESULTS: Cell surface molecules have been identified on colorectal carcinoma cells whose expression appears to be related to malignant transformation, tumor progression, or patient prognosis. Among these cell surface molecules, various cell adhesion molecules, growth factor receptors, proteinases, and their receptors and inhibitors have been identified as potentially useful prognostic markers. CONCLUSIONS: Although data exist on the prognostic values of certain cell surface markers, the use of multivariate analysis for the identification of valuable prognostic factors remains uncommon. Using reproducible and standardized multivariate analysis procedures, new tumor markers should be carefully examined for their biologic and prognostic relevance before being considered as potentially useful in the management of colorectal cancers.

Tumor cell adhesion of human colon carcinoma cells with different metastatic properties to extracellular matrix under dynamic conditions of laminar flow.

PURPOSE: Shear forces have an important influence on cell adhesion and other cellular functions, and malignant cell lines appear to possess different adhesive properties under static and dynamic conditions. Thus, we analyzed human colon carcinoma cell adhesion under dynamic conditions and examined the interactions of HT-29 colon carcinoma cells of different metastatic properties with various immobilized ECM components. METHODS: Wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR), and adhesion stabilization rate (ASR) were compared between the cell lines using dynamic conditions in a laminar flow chamber by decreasing the flow (wall shear stress) of cell suspensions. Patterns of cell adhesion under dynamic conditions were compared to adhesive interactions in static microtiterplate assays. RESULTS: Poorly metastatic HT-29P cells adhered six times more than highly metastatic cells to type I collagen under laminar fluid flow, whereas only highly metastatic HT-29LMM showed adhesive interactions with fibronectin under static and dynamic conditions. High rates of cell adhesion to collagen IV were found under static, but not under dynamic, conditions. CONCLUSIONS: Although poorly and highly metastatic HT-29 cells express similar patterns of integrins, they differ in their adhesive properties to ECM components under static and dynamic conditions. Hydrodynamic shear forces appear to influence adhesive properties of HT-29 cells, and differences between dynamic and static cell adhesion were found.

Partial purification of a liver-derived tumor cell growth inhibitor that differentially inhibits poorly-liver metastasizing cell lines: identification as an active subunit of arginase.

Organ specific tumor metastasis is thought in part to require the ability of metastatic cells to respond to target-organ-associated growth factors or to avoid the effects of target organ associated growth inhibitors. We previously found that murine and rat liver-conditioned media inhibited the growth of the poorly-liver metastasizing murine RAW117-P large-cell lymphoma cells more than their highly liver-metastasizing RAW117-H10 counterparts. Using a six step chromatographic procedure, the major RAW117-P cell proliferation inhibitor from a rat liver extract was purified. The factor displayed a Mr of approximately 35,000 and an isoelectric point > 8.5. This material inhibited the growth of many cells at high concentration; however, in dose-response studies it displayed a higher IC50 for highly-liver metastatic murine RAW117-H10 lymphoma and human KM12SM colon carcinoma cells than for their poorly-liver metastatic counterparts. Attempts to identify the growth inhibitor led to the supplementation of tissue culture inhibitor assays with various components, including excess amino acids, and this was found to completely abrogate the factor's activity. Specifically, the addition of excess arginine resulted in the complete cellular recovery from inhibitor exposure. This tentatively identified the liver growth inhibitor as the enzyme arginase, a Mr approximately 10,000 multisubunit protein. A microtiter plate-based assay for arginase was developed and the purification repeated using human liver as a source of activity and the human KM12C colon carcinoma line as a target. The growth inhibitory and arginase activities were found to co-purify, identifying the factor as arginase. Highly-metastatic cells displayed no ability to preferentially inactivate or inhibit the activity of arginase, but they did they display slightly greater amounts of intracellular arginine. The liver is a major site of arginase localization as the enzyme is required for the functioning of the urea cycle. The results indicate that certain liver-colonizing tumor cells can escape, to a degree, the proliferation-damping effects of arginine depletion.

Downregulation of urokinase-type plasminogen activator receptor (uPAR) induces caspase-mediated cell death in human glioblastoma cells.

Urokinase-type plasminogen activator receptors (uPARs) play an important role in tumor invasion by localizing degradative enzymes at the invasive zone. Our previous studies with human glioblastoma cell line SNB19 expressing AS-uPAR stable tranfectant lose their invasive properties when injected in vivo. The aim of the present study is to investigate whether the inhibition of tumor formation is due to apoptosis. Apoptosis is a highly conserved cell suicide program essential for development and tissue homeostasis of all metazoan organisms. Key to the apoptotic program is a family of cystein proteases termed caspases, which are important for execution of apoptosis by cleavage of essential cellular proteins. We found loss of mitochondrial transmembrane potential, release of cytochrome C from mitochondria and subsequent activation of Caspase-9 in SNB 19 AS-uPAR cells compared to parental and vector controls. Our results indicate that suppression of uPAR results in apoptosis and suggest that Caspase-9 dependent apoptosis plays an important role in SNB19 AS-uPAR-induced apoptosis.

Cell biology and clinical implications of adhesion molecules in colorectal diseases: colorectal cancers, infections and inflammatory bowel diseases.

Adhesion molecules are transmembrane proteins that can anchor cytoskeletal proteins on the cytoplasmic side of the cell membrane, while also connecting extracellular structures on the outer surface of the cell membrane. In addition to physical linkages between the extracellular environment and the cytoskeleton, adhesive complexes participate in important signal transduction systems as modulators or receptors. Their functions in cell signaling are probably at least as important as their cytoskeletal and cell attachment properties. Understanding these regulatory functions appears to be of importance in determining of pathological characteristic of numerous diseases. Expression and functional activity of various adhesion molecules have been found in different diseases affecting the colorectum. In this review we summarize recent advantages about the cell biology these diseases and clinical implications.

Role of tissue factor pathway inhibitor-2 (TFPI-2) in amelanotic melanoma (C-32) invasion.

Human tissue factor pathway inhibitor-2 (TFPI-2), also known as placental protein (PP5) and matrix-associated serine protease inhibitor (MSPI), is a 32-kDa extracellular matrix (ECM) protein consisting of three tandomly arranged Kunitz-type domains that inhibits plasmin, trypsin, chymotrypsin, cathepsin G and plasma kallikrein but not urokinase and tissue-type plasminogen activators or thrombin. Earlier studies in our laboratory revealed that the production of TFPI-2 is reduced or absent during the tumor progression of human gliomas. In the present study, we investigated the role of TFPI-2 in the invasiveness of the amelanotic melanoma cell line C-32. We stably transfected C-32 cells with a vector capable of expressing TFPI-2 in a sense orientation (0.7 kb). TFPI-2 protein production was then determined by western blotting and the mRNA level by northern blotting in parental and stably transfected (vector and sense) clones. The levels of TFPI-2 protein and mRNA were significantly higher in the sense clones, but neither was detected in parental and vector control clones. In addition, in vitro Matrigel invasion/migration assays revealed that the invasive behavior of sense clones was inhibited compared with the behavior of parental and vector clones. This is the first study to show that the upregulation of TFPI-2 plays a significant role in reducing the invasive behavior of human amelanotic melanomas.

Modulation of endothelial cell morphogenesis in vitro by MMP-9 during glial-endothelial cell interactions.

The purpose of this study was to investigate the roles of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the formation of capillary structures by human brain microvascular endothelial cells cocultured with SNB19 glioblastoma cells. Unstimulated cocultures did not form capillaries and produce MMP-9 but stimulation with the protein kinase C (PKC) activator 4-phorbol-12-myristate 13-acetate (PMA) produced MMP-9 and capillary networks. Addition of recombinant MMP-9 increased capillary formation. Anti-MMP-9 antibodies, TIMP-1, the synthetic MMPs inhibitor Batimastat (BB-94), and the PKC inhibitor calphostin-C all reduced MMP-9 activity and capillary network formation in these cocultures. Cytochalasin-D in the presence of PMA suppressed MMP-9 expression and capillary formation, but colchicine-B had no such effect. Finally, PMA-induced MMP-9 expression and capillary formation were inhibited by the MEKK-specific inhibitor PD98059. These results suggest that MMP-9 is important in endothelial cell morphogenesis and the formation of capillaries in glial/endothelial cocultures in vitro.

In vitro modulation of human lung cancer cell line invasiveness by antisense cDNA of tissue factor pathway inhibitor-2.

Human tissue factor pathway inhibitor-2 (TFPI-2) is a Kunitz-type serine protease inhibitor that inhibits plasmin, trypsin, chymotrypsin, cathepsin G and plasma kallikrein but not urokinase (uPA) or tissue-type plasminogen activator and thrombin. Earlier studies from our and other laboratories have shown that the production of TFPI-2 is downregulated during the progression of various cancers. To investigate the role of TFPI-2 in the invasion and metastasis of lung tumors, the human lung cancer cell line A549, which produces high levels of TFPI-2, was stably transfected with a vector capable of expressing an antisense transcript complementary to the full-length TFPI-2 mRNA. Northern blot analysis was used to quantify the TFPI-2 mRNA transcript, and western blot analysis was used to measure TFPI-2 protein levels in parental cells and stably transfected (vector and antisense) clones. The levels of TFPI-2 mRNA and protein were significantly less in antisense clones than in the parental and vector controls. The invasive potential of the parental cells and stably transfected vector clones in vitro, as measured by the Matrigel invasion assay, was also markedly less than that of antisense clones. Further characterization of these clones showed that more cells migrated from antisense clones than from parental and vector clones. These data suggest that TFPI-2 is critical for the invasion and metastasis of lung cancer and that the downregulation of TFPI-2 production may be a feasible approach to increase invasiveness and metastasis.

Regulation of MMP-9 (type IV collagenase) production and invasiveness in gliomas by the extracellular signal-regulated kinase and jun amino-terminal kinase signaling cascades.

Our previous studies have shown that MMP-9 levels are significantly elevated during the progression of human gliomas. In the current study, we examined the role of JNK- and ERK-dependent signaling modules in the regulation of MMP-9 production and the invasive behavior of the human glioblastoma cell line SNB19, in which JNK/ERK1 is constitutively activated. SNB19 cells that were transfected with dominant-negative JNK, MEKK, and ERK1 expression vectors showed reduced MMP-9 promoter activity. In addition, conditioned medium collected from SNB19 cells transfected with these expression vectors showed diminished MMP-9 activity in the presence of phorbol myristate acetate, as determined by gelatin zymography. The cotransfection of SNB19 cells with kinase-deficient c-raf also diminished MMP-9 promoter activity. Further, in the presence of a specific inhibitor of MEKK (PD098059), the Matrigel invasion assay showed the invasiveness of dominant-negative SNB19 cells transfected with dominant-negative JNK1 or ERK1 to be remarkably reduced. In conclusion, our studies showed for the first time that MMP-9 production and the invasive behavior of SNB 19 cells are regulated by JNK- and ERK-dependent signaling modules and that interfering with either of the pathways reduces invasiveness.

Transferrin receptor overexpression enhances transferrin responsiveness and the metastatic growth of a rat mammary adenocarcinoma cell line.

We previously found that breast cancer cell transferrin receptor expression and proliferative response to transferrin often correlated with metastatic capability. To further explore this, we transfected mammary tumor cells with a cDNA coding for the transferrin receptor and examined the effects of its overexpression on various cellular properties. A human transferrin receptor expression plasmid was made by excising the cDNA for the receptor from pcDTR1 and ligating it into the multiple cloning site of pcDNAINeo. The resulting construct was transfected into the poorly metastatic rat MTLn2 line that expresses low endogenous levels of rat transferrin receptor, and transfection-induced receptor expression was ascertained using antibodies specific for the human protein. Approximately 50% of the initial geneticin-resistant transfected MTLn2 cells overexpressed human transferrin receptor protein. High expressors were further isolated by four sequential FACS sorts. The final cell population expressed approximately 3-7 times more cell surface transferrin receptor than did vector transfected controls. Both lines proliferated at the same rate in normal (medium plus 5% FBS) culture conditions. However, in serum-free conditions, the transferrin receptor overexpressor cells displayed a pronounced proliferative response to transferrin whereas the control line did not. When injected into the mammary fat pads of female nude mice, cells from both lines formed micrometastases to the lung that were specifically visualized by immunohistochemical staining of rat cytokeratin 17. This revealed that the transferrin receptor transfected line formed larger lesions of this nature than did cells from the vector transfected controls. When injected into the tail vein of female nude mice, the transferrin receptor overexpressors likewise formed gross lung metastases of remarkably greater size than did the vector only transfectants. Overexpression of cell surface human transferrin receptor on MTLn2 cells appeared to affect their in vitro growth response to transferrin and their ability to grow at a secondary site in vivo.

Different adhesion properties of highly and poorly metastatic HT-29 colon carcinoma cells with extracellular matrix components: role of integrin expression and cytoskeletal components.

Integrin-mediated tumour cell adhesion to extracellular matrix (ECM) components is an important step in the development of metastatic lesions. Thus, integrin expression and integrin-mediated adhesion of colon carcinoma cells to various ECM components was examined. Poorly (HT-29P) and highly (HT-29LMM) liver-metastatic colon carcinoma cells were used to study the rates of adhesion to collagen I (C I), collagen IV (C IV), laminin (LN), fibronectin (FN), or vitronectin (VN) in a static adhesion assay (10-120 min). Cells were untreated or treated with oligopeptides (RGD, GRGDS, YIGSR, RGES), anti-integrin antibodies, or colchicine, nocodazole, cycloheximide, acrylamide or cytochalasin D (to disrupt cytoskeletal structures). Both cell lines expressed similar patterns of integrin expression (alpha2, alpha3, ,alpha6, alphav, beta1, beta4, and beta5) by immunocytochemistry and immunoprecipitation. HT-29LMM cells showed significantly higher rates of adhesion to LN (P < 0.001) and FN (P < 0.001), but significantly poorer rates of adhesion to C I (P < 0.05) and C IV (P < 0.001) than HT-29P cells, respectively, adhesion to VN was insignificant. RGD and GRGDS inhibited HT-29LMM cell adhesion to FN only. Pretreatment with anti-beta, or anti-alpha2 integrin subunits suppressed adhesion to C I and C IV, and adhesion to LN was inhibited with anti-beta1 or anti-alpha6 integrin. Anti-beta1 or anti-alphav blocked adhesion to FN. Pretreatment of cells with cytochalasin D, cycloheximide or acrylamide inhibited adhesive interactions of both cell lines to the ECM components. In contrast, colchicine and nocodazole had no effect. The results demonstrate that adhesion of HT-29 cells to ECM is mediated, in part, by different integrins, depending on the substrate. Poorly and highly metastatic HT-29 cells possessed different patterns of adhesion to the various ECM substrates, but these differences were not due to different expression of integrin subunits. The results also suggested that the initial adhesion of poorly or highly metastatic HT-29 cells to ECM components requires, in part, the presence of native action and intermediate filaments, but not of microtubules. Thus the adhesion of tumour cells to ECM components may be dependent on signal transduction and assembly of microfilaments.