The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells

We have previously isolated a series of MCF-7 human breast cancer cell variants which no longer require estrogen-supplementation for tumor growth in nude mice (Clarke et al. Proc Natl Acad Sci USA 86: 3649-3653, 1989). We now report that these hormone-independent and hormone-responsive variants (MIII, MCF7/LCC1) can invade locally from solid mammary fat pad tumors, and produce primary extensions on the surface of intraperitoneal structures including liver, pancreas, and diaphragm. Both lymphatic and hematogenous dissemination are observed, resulting in the establishing of pulmonary, bone, and renal metastases. The pattern of metastasis by MIII and MCF7/LCC1 cells closely resembles that frequently observed in breast cancer patients, and provides the first evidence of metastasis from MCF-7 cells growing in vivo without supplementary estrogen. The interexperimental incidence of metastases, and the time from cell inoculation to the appearance of metastatic disease are variable. The increased metastatic potential is not associated with an increase in either the level of laminin attachment, laminin receptor mRNA expression, or secreted type IV collagenolytic activity. We also did not detect a significant decrease in the steady-state mRNA levels of the metastasis inhibitor nm23 gene. However, when growing without estrogen in vitro, MCF7/LCC1 cells produce elevated levels of the estrogen-inducible cathepsin D enzyme.

Upregulated MT1-MMP/TIMP-2 axis in the TSU-Pr1-B1/B2 model of metastatic progression in transitional cell carcinoma of the bladder

Muscle invasive transitional cell carcinoma (TCC) of the bladder is associated with a high frequency of metastasis, resulting in poor prognosis for patients presenting with this disease. Models that capture and demonstrate step-wise enhancement of elements of the human metastatic cascade on a similar genetic background are useful research tools. We have utilized the transitional cell carcinoma cell line TSU-Pr1 to develop an in vivo experimental model of bladder TCC metastasis. TSU-Pr1 cells were inoculated into the left cardiac ventricle of SCID mice and the development of bone metastases was monitored using high resolution X-ray. Tumor tissue from a single bone lesion was excised and cultured in vitro to generate the TSU-Pr1-B1 subline. This cycle was repeated with the TSU-Pr1-B1 cells to generate the successive subline TSU-Pr1-B2. DNA profiling and karyotype analysis confirmed the genetic relationship of these three cell lines. In vitro, the growth rate of these cell lines was not significantly different. However, following intracardiac inoculation TSU-Pr1, TSU-Pr1-B1 and TSU-Pr1-B2 exhibited increasing metastatic potential with a concomitant decrease in time to the onset of radiologically detectable metastatic bone lesions. Significant elevations in the levels of mRNA expression of the matrix metalloproteases (MMPs) membrane type 1-MMP (MT1-MMP), MT2-MMP and MMP-9, and their inhibitor, tissue inhibitor of metalloprotease-2 (TIMP-2), across the progressively metastatic cell lines, were detected by quantitative PCR. Given the role of MT1-MMP and TIMP-2 in MMP-2 activation, and the upregulation of MMP-9, these data suggest an important role for matrix remodeling, particularly basement membrane, in this progression. The TSU-Pr1-B1/B2 model holds promise for further identification of important molecules.

Invasive and metastatic properties of MCF-7 cells and ras(H)-transfected MCF-7 cell lines

In vitro invasion and in vivo metastasis assays were performed with a panel of MCF-7 cells transfected with isogenic constructs of mutated ras(H) genes. Both increased levels of ras(H) expression and ras(H) oncogene activation increased activity of derivative cell lines in in vitro invasion assays. In vivo formation of spontaneous metastases was assessed after intradermal inoculation of MCF-7 cells in the vicinity of the mammary fat pads of ovariectomized nude mice. No metastases were seen in the absence of estradiol treatment of the mice. With estradiol supplementation of the mice both the ras(H)-transfected and control transfected cell lines gave a higher incidence of metastases than parental MCF-7 cells. Prolonged treatment of mice with exogenous estradiol (60 days vs. 21 days) resulted in more frequent metastases to liver and lung at the end of the 90-day observation period. In contrast to activated ras(H)-gene enhancement of metastatic capacity of rodent fibroblast and epithelial cell lines, there was no correlation of ras(H) expression with in vivo metastatic capacity of a human mammary carcinoma cell line.

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by αVβ3 integrin in human breast cancer cells

The influence of αVβ3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing β3 integrin status. Overexpression of β3 integrin caused increased cell surface expression of αV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. β3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, αVβ3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of β3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with β3 integrin expression. Although our studies confirm important biological effects of αVβ3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, β3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by αVβ3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.

Interrupting malaria transmission: quantifying the impact of interventions in regions of low to moderate transmission

Malaria has been eliminated from over 40 countries with an additional 39 currently planning for, or committed to, elimination. Information on the likely impact of available interventions, and the required time, is urgently needed to help plan resource allocation. Mathematical modelling has been used to investigate the impact of various interventions; the strength of the conclusions is boosted when several models with differing formulation produce similar data. Here we predict by using an individual-based stochastic simulation model of seasonal Plasmodium falciparum transmission that transmission can be interrupted and parasite reintroductions controlled in villages of 1,000 individuals where the entomological inoculation rate is <7 infectious bites per person per year using chemotherapy and bed net strategies. Above this transmission intensity bed nets and symptomatic treatment alone were not sufficient to interrupt transmission and control the importation of malaria for at least 150 days. Our model results suggest that 1) stochastic events impact the likelihood of successfully interrupting transmission with large variability in the times required, 2) the relative reduction in morbidity caused by the interventions were age-group specific, changing over time, and 3) the post-intervention changes in morbidity were larger than the corresponding impact on transmission. These results generally agree with the conclusions from previously published models. However the model also predicted changes in parasite population structure as a result of improved treatment of symptomatic individuals; the survival probability of introduced parasites reduced leading to an increase in the prevalence of sub-patent infections in semi-immune individuals. This novel finding requires further investigation in the field because, if confirmed, such a change would have a negative impact on attempts to eliminate the disease from areas of moderate transmission.

Experimental colonization of the canine urinary tract with the asymptomatic bacteriuria Escherichia coli strain 83972

Establishment of asymptomatic bacteriuria (ABU) with Escherichia coli 83972 is a viable prophylactic alternative to antibiotic therapy for the prevention of recurrent bacterial urinary tract infection in humans. Approximately 2 x 108 viable E. coli 83972 cells were introduced into the bladder of six healthy female dogs via a sterile urinary catheter. The presence of pyuria, depression, stranguria, pollakiuria and haematuria was documented for 6 weeks and urinalysis and aerobic bacterial cultures were performed every 24–72 h. Pyuria was present in all dogs on day 1 post-inoculation and 4/6 dogs (67%) had a positive urine culture on this day. Duration of colonization ranged from 0 to 10 days (median 4 days). Four dogs were re-inoculated on day 20. Duration of colonization following the second inoculation ranged from 1 to 3 days. No dog suffered pyrexia or appeared systemically unwell but all dogs initially exhibited mild pollakiuria and a small number displayed gross haematuria and/or stranguria. By day 3 of each trial all clinical signs had resolved. Persistent bacteriuria was not achieved in any dog but two dogs were colonized for 10 days following a single inoculation. Further research is required to determine whether establishment of ABU in dogs with recurrent urinary tract infection is a viable alternative to repeated doses of antimicrobial agents.

The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events

Background: Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. Methods: Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector - target cell contact and adsorption periods were studied. MOI between 0-32 was assessed on commonly used cell lines as well as a new cell line. Results: We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0-32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. Conclusion: Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted. © 2004 Zhang et al; licensee BioMed Central Ltd.

Plant height affects Fusarium crown rot severity in wheat

Effects of plant height on Fusarium crown rot (FCR) disease severity were investigated using 12 pairs of near-isogenic lines (NILs) for six different reduced height (Rht) genes in wheat. The dwarf isolines all gave better FCR resistance when compared with their respective tall counterparts, although the Rht genes involved in these NILs are located on several different chromosomes. Treating plants with exogenous gibberellin increased FCR severity as well as seedling lengths in all of the isolines tested. Analysis of the expression of several defense genes with known correlation with resistance to FCR pathogens between the Rht isolines following FCR inoculation indicated that the better resistance of the dwarf isolines was not due to enhanced defense gene induction. These results suggested that the difference in FCR severity between the tall and dwarf isolines is likely due to their height difference per se or to some physiological and structural consequences of reduced height. Thus, caution should be taken when considering to exploit any FCR locus located near a height gene.

Rhizoctonia crown and root rot of the pasture legume, sulla (Hedysarum coronarium)

Rhizoctonia solani AG-2-2 was isolated from wilting and dying plants of sulla (Hedysarum coronarium), which is currently being assessed in eastern and southern Australia for its potential as a pasture and forage legume. Infected plants in the field had extensive rotting of the taproot, lateral roots and crown. Koch's postulates were fulfilled using three inoculation methods. The disease may pose a considerable threat to the potential use of H. coronarium in the dryland, grazing farming systems of Australia, with resistance offering the most viable option for minimising its impact.

Selection and characterisation of two attenuated vaccine lines of Eimeria tenella in Australia

Objective To attenuate two strains of Eimeria tenella by selecting for precocious development and evaluate the strains in characterisation trials and by field evaluation, to choose one precocious line for incorporation into an Australian live coccidiosis vaccine for poultry. Design Two strains from non-commercial flocks were passaged through chickens while selecting for precocious development. Each strain was characterised for drug sensitivity, pathogenicity, protection against homologous and heterologous challenge, and oocyst output in replicated experiments in which the experimental unit was a cage of three birds. Oocyst output and/or body weight gain data collected over a 10 to 12 day period following final inoculation were measured. Feed conversion ratios were also calculated where possible. Results Fifteen passages resulted in prepatent periods reduced by 24 h for the Redlands strain (from 144 h to 120 h)and 23 h for the Darryl strain (from 139 h to 116 h). Characterisation trials demonstrated that each precocious line was significantly less pathogenic than its parent strain and each effectively induced immunity that protected chickens against challenge with both the parent strain and other virulent field strains. Both lines had oocyst outputs that, although significantly reduced relative to the parent strains, remained sufficiently high for commercial vaccine production, and both showed susceptibility to coccidiostats. Conclusion Two attenuated lines have been produced that exhibit the appropriate characteristics for use in an Australian live coccidiosis vaccine.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Development of a small-plant bioassay to assess banana grown from tissue culture for consistent infection by Fusarium oxysporum f. sp. cubense

Two reliable small-plant bioassays were developed using tissue-cultured banana, resulting in consistent symptom expression and infection by Fusarium oxysporum f. sp. cubense (Foc). One bioassay was based on providing a constant watertable within a closed pot and the second used free-draining pots. Culture medium for spore generation influenced infectivity of Foc. Inoculation of potted banana by drenching potting mix with a conidial suspension, consisting mostly of microconidia, few macroconidia and no chlamydospores, generated from one-quarter-strength potato dextrose agar + streptomycin sulfate, resulted in inconsistent infection. When a conidial suspension that consisted of all three spore types, microconidia, macroconidia and chlamydospores, prepared from spores generated on carnation leaf agar was used, all plants became infected, indicating that the spore type present in conidial suspensions may contribute to inconsistency of infection. Inconsistency of infection was not due to loss of virulence of the pathogen in culture. Millet grain precolonised by Foc as a source of inoculum resulted in consistent infection between replicate plants. Sorghum was not a suitable grain for preparation of inoculum as it was observed to discolour roots and has the potential to stunt root growth, possibly due to the release of phytotoxins. For the modified closed-pot system, a pasteurised potting mix consisting of equal parts of bedding sand, perlite and vermiculite plus 1 g/L Triabon slow release fertiliser was suitable for plant growth and promoted capillary movement of water through the potting mix profile. A suitable potting mix for the free-draining pot system was also developed.

Concentrations of constitutive alk(en)ylresorcinols in peel of commercial mango varieties and resistance to postharvest anthracnose

Mature green mango fruits of commercially important varieties were screened to investigate the levels of constitutive antifungal compounds in peel and to assess anthracnose disease after inoculation with Colletotrichum gloeosporioides. High pressure liquid chromatography was used to quantify the levels of 5-n-heptadecenylresorcinol and 5-n-pentadecylresorcinol in the peel extracts. The fruit peel of the varieties ‘Kensington Pride’ and ‘Keitt’ were observed to have the highest levels of both 5-n-heptadecenylresorcinol (107.3-123.7 and 49.9-61.4 μg/g FW, respectively) and 5-n-pentadecylresorcinol (6.32-7.99 and 3.30-6.05 μg/g FW, respectively), and the fruit of the two varieties were found to have some resistance to postharvest anthracnose. The varieties ‘Kent’, ‘R2E2’, ‘Nam Doc Mai’, ‘Calypso’, and ‘Honey Gold’ contained much lower concentrations of resorcinols in their peel and three of these varieties were found to be more susceptible to anthracnose. Concentrations of 5-nheptadecenylresorcinol were significantly lower at the ‘sprung’ and ‘eating ripe’ stages of ripening compared to levels at harvest. Concentrations of 5-n-pentadecylresorcinol did not differ significantly across the three stages of ripening. The levels of these two resorcinols were found to be strongly inter-correlated (P < 0.001, r2 = 0.71), with concentrations of 5-nheptadecenylresorcinol being an average 18 times higher than those of 5-npentadecylresorcinol. At the ‘eating ripe’ stage, significant relationships were observed between the concentrations of each type of alk(en)ylresorcinol and anthracnose lesion areas following postharvest inoculation, P<0.001, r2= 0.69 for 5-n pentadecylresorcinol, and P<0.001, r2= 0.44 for 5-n-heptadecenylresorcinol.

Infection, colonisation and sporulation by Pseudocercospora macadamiae on macadamia fruit

The structures and manner with which Pseudocercospora macadamiae penetrates, colonises and proliferates from the pericarp of macadamia fruit was studied using scanning electron microscopy and fluorescence light microscopy. Germ tubes arising from conidia penetrated open stomata within 20 h of inoculation, without observation of specialised infection structures such as appressoria. Colonisation of the pericarp was intercellular, without observation of specialised intracellular infection structures such as haustoria, and was complete from the epidermis to the mesocarp. The fungus proliferated at the epidermis by the formation of conidiophores and conidia on substomatal and protuberant subepidermal stromata. These structures were not observed on the mesocarp surface. The onset of visual husk spot symptoms coincided with an increase in pathogen biomass on the pericarp surface. The progression of symptoms from tan-coloured spots to larger red-brown lesions coincided with the production of conidiophores from substomatal and protuberant subepidermal stromata. The darker the colour of the husk spot lesion, the more frequently protuberant subepidermal stromata were observed. These findings are discussed in the context of observation of other cercosporoid fungi.