Establishment and characterization of two human breast carcinoma cell lines by spontaneous immortalization: Discordance between Estrogen, Progesterone and HER2/neu receptors of breast carcinoma tissues with derived cell lines

Background

Breast cancer is one of the most common cancers among women throughout the world. Therefore, established cell lines are widely used as in vitro experimental models in cancer research.

Methods

Two continuous human breast cell lines, designated MBC1 and MBC2, were successfully established and characterized from invasive ductal breast carcinoma tissues of Malaysian patients. MBC1 and MBC2 have been characterized in terms of morphology analysis, population doubling time, clonogenic formation, wound healing assay, invasion assay, cell cycle, DNA profiling, fluorescence immunocytochemistry, Western blotting and karyotyping.

Results

MBC1 and MBC2 exhibited adherent monolayer epithelial morphology at a passage number of 150. Receptor status of MBC1 and MBC2 show (ER⁺, PR⁺, HER2⁺) and (ER⁺, PR^-, HER2⁺), respectively. These results are in discordance with histopathological studies of the tumoral tissues, which were triple negative and (ER^-, PR^-, HER2⁺) for MBC1 and MBC2, respectively. Both cell lines were capable of growing in soft agar culture, which suggests their metastatic potential. The MBC1 and MBC2 metaphase spreads showed an abnormal karyotype, including hyperdiploidy and complex rearrangements with modes of 52–58 chromosomes per cell.

Conclusions

Loss or gain in secondary properties, deregulation and specific genetic changes possibly conferred receptor changes during the culturing of tumoral cells. Thus, we hypothesize that, among heterogenous tumoral cells, only a small minority of ER⁺/PR⁺/HER2⁺ and ER⁺/PR^-/HER2⁺ cells with lower energy metabolism might survive and adjust easily to in vitro conditions. These cell lines will pave the way for new perspectives in genetic and biological investigations, drug resistance and chemotherapy studies, and would serve as prototype models in Malaysian breast carcinogenesis investigations.     

Biotechnological route for sustainable succinate production utilizing oil palm frond and kenaf as potential carbon sources

Applied Microbiology and Biotechnology - Due to the world’s dwindling energy supplies, greater thrust has been placed on the utilization of renewable resources for global succinate...     

Site-saturation mutagenesis of mutant <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase II signal peptide hydrophobic region for improved excretion of cyclodextrin glucanotransferase

The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.     

Temporary sterilization behavior of mutualistic partner ants in a Southeast Asian myrmecophyte

Obligate ant–plant interactions are known to be mutualistic but plant-ants that destroy flowers of their hosts have been reported. They were regarded as parasites in myrmecophytic systems. The mechanisms that lead to flower damage (sterilization) by plant-ants are not easy to understand as most sterilizing ants are actually regular colonizers of their plants and normally offer protection against herbivores and/or plant competition. It is difficult to find general patterns of ant or plant traits even in the few yet known associations of flower sterilization. We here present the first study from Southeast Asia where flower sterilizing occurs in the complex mutualistic Macaranga–Crematogaster system that differs from other cases. Flowers of M. hullettii in the Gombak Valley were destroyed by all three associated specific and otherwise protective Crematogaster species. The hypotheses that limitation of nesting space or food are main proximate factors for flower destruction were not strongly supported in our study system. Ants are even attracted to flowers by special food bodies produced by the plants. Only younger, not yet reproductive colonies were found to destroy flowers but not colonies with alates, indicating that flower sterilization behavior may only occur when the onset of host reproduction precedes ant reproduction, perhaps leading to a change in ant behavior. Fruit set always occurred in larger trees, and saplings for colonizing ant queens were therefore always present in the local population, stabilizing the association.     

1α,25(OH)2D3 inhibits FGF-2 release from oral squamous cell carcinoma cells through down-regulation of HBp17/FGFBP-1

Heparin-binding protein 17/fibroblast growth factor binding protein-1 (HBp17/FGFBP-1, GenBank accession no. NP-005121) is prominent for its role as the chaperone for fibroblast growth factor-2 (FGF-2), which plays a crucial role in angiogenesis as well as promoting tumor growth. HBp17/FGFBP-1 has been proposed as a candidate biomarker for a number of cancers since it is frequently found to be elevated in many cancer types including in the tissue and cell lines of oral squamous cell carcinomas (OSCC). Previously, we reported that 1α,25(OH)₂D₃ suppressed the HBp17/FGFBP-1 expression in OSCC by inhibiting nuclear factor-kappaB (NF-κB) expression via vitamin D3 receptor (VDR). In this paper, to further characterize the inhibitory effect of 1α,25(OH)₂D₃ on HBp17/FGFBP-1, we examined the cellular localization of HBp17/FGFBP-1 protein and FGF-2 protein in the UE OSCC cell line. We found that the treatment of OSCC cells with 40-nM 1α,25(OH)₂D₃ suppressed HBp17/FGFBP-1 expression both in the nucleus and cytosol and reduced FGF-2 release into the culture medium. The expression of HBp17/FGFBP-1 and FGF-2 was analyzed by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). In summary, the ability of 1α,25(OH)₂D₃ to suppress the expression of HBp17/FGFBP-1 and FGF-2 strongly suggests a therapeutic potential as a molecular-targeted anticancer drug for FGF-dependent cancers.     

Novel synthetic signal peptides for the periplasmic secretion of green fluorescent protein in Escherichia coli

New secretion vectors containing synthetic signal peptides were constructed to study the periplasmic translocation of green fluorescent protein (GFP) in Escherichia coli. These constructs encode synthetic signal peptides spA and spD fused to the amino terminal end of GFP, and expressed from T7/lac promoter in the BL21DE3 strain by induction with IPTG. The recombinant protein was detected in both the cytoplasmic and periplasmic fractions. Fluorescence analysis revealed that recombinant proteins with signal peptides were not fluorescent, indicating translocation to periplasmic space. In contrast, recombinant proteins without signal peptide were fluorescent. These results indicate that the expressed recombinant proteins were translocated into the periplasm. Therefore, the synthetic signal peptides derived from signal peptides of Bacillus sp. could efficiently secrete the heterologous proteins to the periplasmic space of E. coli.     

Simplified feeding strategies for the fed-batch cultivation of Kluyveromyces lactis GG799 for enhanced recombinant xylanase production

A xylanase gene (xyn2) from Trichoderma reesei ATCC 58350 was previously cloned and expressed in Kluyveromyces lactis GG799. The production of the recombinant xylanase was conducted in a developed medium with an optimised batch and with fed-batches that were processed with glucose. The glucose served as a carbon source for cell growth and as an inducer for xylanase production. In a 1-L batch system, a glucose concentration of 20 g L^?1 and 80 % dissolved oxygen were found to provide the best conditions for the tested ranges. A xylanase activity of 75.53 U mL^?1 was obtained. However, in the batch mode, glucose depletions reduced the synthesis of recombinant xylanase by K. lactis GG799. To maximise the production of xylanase, further optimisation was performed using exponential feeding. We investigated the effects of various nitrogen sources combined with the carbon to nitrogen (C/N) molar ratio on the production of xylanase. Of the various nitrogen sources, yeast extract was found to be the most useful for recombinant xylanase production. The highest xylanase production (110.13 U mL^?1) was measured at a C/N ratio of 50.08. These conditions led to a 45.8 % increase in xylanase activity compared with the batch cultures. Interestingly, the further addition of 500 g L^?1 glucose led to a 6.2-fold increase (465.07 U mL^?1) in recombinant xylanase activity. These findings, together with those of the exponential feeding strategy, indicate that the composition of the C/N molar ratio has a substantial impact on recombinant protein production in K. lactis.