Bioactive diterpenoid metabolism and cytotoxic activities of genetically transformed Euphorbia lathyris roots.

Plants in the genus Euphorbia produce a wide variety of pharmacologically active diterpenoids with anticancer, multidrug resistance reversal, and antiviral properties. Some are the primary industrial source of ingenol mebutate, which is approved for treatment of the precancerous skin condition actinic keratosis. Similar to other high value phytochemicals, Euphorbia diterpenoids accumulate at low concentrations in planta and chemical synthesis produces similarly low yields. We established genetically transformed root cultures of Euphorbia lathryis as a strategy to gain greater access to diterpenoids from this genus. Transformed roots produced via stem explant infection with Agrobacterium rhizogenes strain 15834 recapitulated the metabolite profiles of field-grown plant roots and aerial tissues. Several putative diterpenoids were present in transformed roots, including ingenol and closely related structures, indicating that root cultures are a promising approach to Euphorbia-specific diterpenoid production. Treatment with methyl jasmonate led to a significant, albeit transient increase in mRNA levels of early diterpenoid biosynthetic enzymes (farnesyl pyrophosphate synthase, geranylgeranyl pyrophosphate synthase, and casbene synthase), suggesting that elicitation could prove useful in future pathway characterization and metabolic engineering efforts. We also show the potential of transformed E. lathyris root cultures for natural product drug discovery applications by measuring their cytotoxic activities using a panel of human carcinoma cell lines derived from prostate, cervix, breast, and lung.

Generation of a Full-Thickness Human Skin Equivalent on an Immunodeficient Mouse.

Human skin equivalents composed of epidermal cells and fibroblasts are important for modeling human epidermal development, testing new therapeutics, and designing novel treatment strategies for human skin diseases. Here, we describe a procedure for the generation of an in vivo full-thickness human skin equivalent on an immunodeficient mouse using a grafting chamber system. The protocol involves mixing human epidermal cells and fibroblasts in a silicone grafting chamber that is surgically inserted onto the muscle fascia of a recipient immunodeficient mouse. Following the removal of the silicone chamber, the graft area is exposed to air to induce stratification of developing epidermis, resulting in the reconstitution of full-thickness human skin tissue on a live mouse. This grafting system provides a straightforward approach to study human skin diseases in an animal model and has been previously used to determine the ability of both mouse and human primary epidermal cells and cells derived from pluripotent stem cells to regenerate functional skin in vivo.

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Induction of proto-oncogene BRF2 in breast cancer cells by the dietary soybean isoflavone daidzein.

BACKGROUND: BRF2 is a transcription factor required for synthesis of a small group of non-coding RNAs by RNA polymerase III. Overexpression of BRF2 can transform human mammary epithelial cells. In both breast and lung cancers, the BRF2 gene is amplified and overexpressed and may serve as an oncogenic driver. Furthermore, elevated BRF2 can be independently prognostic of unfavorable survival. Dietary soy isoflavones increase metastasis to lungs in a model of breast cancer and a recent study reported significantly increased cell proliferation in breast cancer patients who used soy supplementation. The soy isoflavone daidzein is a major food-derived phytoestrogen that is structurally similar to estrogen. The putative estrogenic effect of soy raises concern that high consumption of soy foods by breast cancer patients may increase tumor growth. METHODS: Expression of BRF2 RNA and protein was assayed in ER-positive or -negative human breast cancer cells after exposure to daidzein. We also measured mRNA stability, promoter methylation and response to the demethylating agent 5-azacytidine. In addition, expression was compared between mice fed diets enriched or deprived of isoflavones. RESULTS: We demonstrate that the soy isoflavone daidzein specifically stimulates expression of BRF2 in ER-positive breast cancer cells, as well as the related factor BRF1. Induction is accompanied by increased levels of non-coding RNAs that are regulated by BRF2 and BRF1. Daidzein treatment stabilizes BRF2 and BRF1 mRNAs and selectively decreases methylation of the BRF2 promoter. Functional significance of demethylation is supported by induction of BRF2 by the methyltransferase inhibitor 5-azacytidine. None of these effects are observed in an ER-negative breast cancer line, when tested in parallel with ER-positive breast cancer cells. In vivo relevance is suggested by the significantly elevated levels of BRF2 mRNA detected in female mice fed a high-isoflavone commercial diet. In striking contrast, BRF2 and BRF1 mRNA levels are suppressed in matched male mice fed the same isoflavone-enriched diet. CONCLUSIONS: The BRF2 gene that is implicated in cancer can be induced in human breast cancer cells by the isoflavone daidzein, through promoter demethylation and/or mRNA stabilization. Dietary isoflavones may also induce BRF2 in female mice, whereas the converse occurs in males.