3, 3′-diindolylmethane Enhances the Effectiveness of Herceptin against HER-2/Neu-Expressing Breast Cancer Cells

Herceptin failure is a major clinical problem in breast cancer. A subset of breast cancer patients with high HER-2/neu levels eventually experience metastatic disease progression when treated with Herceptin as a single agent. Mechanistic details of development of this aggressive disease are not clear. Therefore, there is a dire need to better understand the mechanisms by which drug resistance develops and to design new combined treatments that benefit patients with aggressive breast cancer and have minimal toxicity. We hypothesized that 3, 3′-diindolylmethane (DIM), a non-toxic agent can be combined with Herceptin to treat breast cancers with high levels of HER-2/neu. Here, we evaluated the effects of Herceptin alone and in combination with DIM on cell viability, apoptosis and clonogenic assays in SKBR3 (HER-2/neu-expressing) and MDA-MB-468 (HER-2/neu negative) breast cancer cells. We found that DIM could enhance the effectiveness of Herceptin by significantly reducing cell viability, which was associated with apoptosis-induction and significant inhibition of colony formation, compared with single agent treatment. These results were consistent with the down-regulation of Akt and NF-kB p65. Mechanistic investigations revealed a significant upregulation of miR-200 and reduction of FoxM1 expression in DIM and Herceptin-treated breast cancer cells. We, therefore, transfected cells with pre-miR-200 or silenced FoxM1 in these cells for understanding the molecular mechanism involved. These results provide experimental evidence, for the first time, that DIM plus Herceptin therapy could be translated to the clinic as a therapeutic modality to improve treatment outcome of patients with breast cancer, particularly for the patients whose tumors express high levels of HER-2/neu.     

Does Auxin Binding Protein 1 Control Both Cell Division and Cell Expansion?

The Auxin-Binding Protein 1 (ABP1) was identified over 30 years ago thanks to it''s high affinity for active auxins. ABP1 plays an essential role in plant life yet to this day, its function remains ‘enigmatic.’ A recent study by our laboratory shows that ABP1 is critical for regulation of the cell cycle, acting both in G₁ and at the G₂/M transition. We showed that ABP1 is likely to mediate the permissive auxin signal for entry into the cell cycle. These data were obtained by studying a conditional functional knock-out of ABP1 generated by cellular immunization in the model tobacco cell line, Bright Yellow 2.Key Words: auxin responses, auxin-binding protein 1, immunomodulation, cellular immunisation     

Molecular systematics and paleobiogeography of the South American sigmodontine rodents [published erratum appears in Mol Biol Evol 1998 Feb;15(2):224]

The murid rodent subfamily Sigmodontinae contains 79 genera which are distributed throughout the New World. The time of arrival of the first sigmodontines in South America and the estimated divergence time(s) of the different lineages of South American sigmodontines have been controversial due to the lack of a good fossil record and the immense number of extant species. The "early-arrival hypothesis" states that the sigmodontines must have arrived in South America no later than the early Miocene, at least 20 MYA, in order to account for their vast present-day diversity, whereas the "late-arrival hypothesis" includes the sigmodontines as part of the Plio-Pleistocene Great American Interchange, which occurred approximately 3.5 MYA. The phylogenetic relationships among 33 of these genera were reconstructed using mitochondrial DNA (mtDNA) sequence data from the ND3, ND4L, arginine tRNA, and ND4 genes, which we show to be evolving at the same rate. A molecular clock was calibrated for these genes using published fossil dates, and the genetic distances were estimated from the DNA sequences in this study. The molecular clock was used to estimate the dates of the South American sigmodontine origin and the main sigmodontine radiation in order to evaluate the "early-" and "late-arrival" scenarios. We estimate the time of the sigmodontine invasion of South America as between approximately 5 and 9 MYA, supporting neither of the scenarios but suggesting two possible models in which the invading lineage was either (1) ancestral to the oryzomyines, akodonts, and phyllotines or (2) ancestral to the akodonts and phyllotines and accompanied by the oryzomyines. The sigmodontine invasion of South America provides an example of the advantage afforded to a lineage by the fortuitous invasion of a previously unexploited habitat, in this case an entire continent.      

Multiple duplications of yeast hexose transport genes in response to selection in a glucose-limited environment

When microbes evolve in a continuous, nutrient-limited environment, natural selection can be predicted to favor genetic changes that give cells greater access to limiting substrate. We analyzed a population of baker's yeast that underwent 450 generations of glucose-limited growth. Relative to the strain used as the inoculum, the predominant cell type at the end of this experiment sustains growth at significantly lower steady-state glucose concentrations and demonstrates markedly enhanced cell yield per mole glucose, significantly enhanced high-affinity glucose transport, and greater relative fitness in pairwise competition. These changes are correlated with increased levels of mRNA hybridizing to probe generated from the hexose transport locus HXT6. Further analysis of the evolved strain reveals the existence of multiple tandem duplications involving two highly similar, high- affinity hexose transport loci, HXT6 and HXT7. Selection appears to have favored changes that result in the formation of more than three chimeric genes derived from the upstream promoter of the HXT7 gene and the coding sequence of HXT6. We propose a genetic mechanism to account for these changes and speculate as to their adaptive significance in the context of gene duplication as a common response of microorganisms to nutrient limitation.      

Unicellular, aerobic nitrogen-fixing cyanobacteria of the genus Cyanothece.

Two marine, unicellular aerobic nitrogen-fixing cyanobacteria, Cyanothece strain BH63 and Cyanothece strain BH68, were isolated from the intertidal sands of the Texas Gulf coast in enrichment conditions designed to favor rapid growth. By cell morphology, ultrastructure, a GC content of 40%, and aerobic nitrogen fixation ability, these strains were assigned to the genus Cyanothece. These strains can use molecular nitrogen as the sole nitrogen source and are capable of photoheterotrophic growth in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and glycerol. The strains demonstrated a doubling time of 10 to 14 h in the presence of nitrate and 16 to 20 h under nitrogen-fixing conditions. Rapid growth of nitrogen-fixing cultures can be obtained in continuous light even when the cultures are continuously shaken or bubbled with air. Under 12-h alternating light and dark cycles, the aerobic nitrogenase activity was confined to the dark phase. The typical rates of aerobic nitrogenase activity in Cyanothece strains BH63 and BH68 were 1,140 and 1,097 nmol of C2H2 reduced per mg (dry weight) per h, respectively, and nitrogenase activity was stimulated twofold by light. Ultrastructural observations revealed that numerous inclusion granules formed between the photosynthetic membranes in cells grown under nitrogen-fixing conditions. These Cyanothece strains posses many characteristics that make them particularly attractive for a detailed analysis of the interaction of nitrogen fixation and photosynthesis in an aerobic diazotroph.     

Efficient liver-specific deletion of a floxed human angiotensinogen transgene by adenoviral delivery of Cre recombinase in vivo.

Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.