Abhijeet Kate, Arpana G Panicker, Diana Writer, Giridharan P, Keshav K V Ramamoorthy, Saji George, Shailendra K Sonawane
Protoplast fusion and transformation: A tool for activation of latent gene clusters
In the quest to discover new bioactive leads for unmet medical needs, actinomycetes present a treasure trove of undiscovered molecules. The ability of actinomycetes to produce antibiotics and other bioactive secondary metabolites has been underestimated due to sparse studies of cryptic gene clusters. These gene clusters can be tapped to explore scaffolds hidden in them. The up-regulation of the dormant genes is one of the most important areas of interest in the bioactive compounds discovery from microbial resources. Genome shuffling is a powerful tool for the activation of such gene clusters. Lei Yu, et al.1, reported enhancement of the lactic acid production in Lactobacillus rhamnosus through genome shuffling brought about by protoplast fusion. D. A. Hopwood et al.2 suggested that an interspecific recombination between strains producing different secondary metabolites, generate producers of ‘hybrid’ antibiotics. They also mentioned that an intraspecific fusion of actinomycetes protoplast bring about random and high frequency recombination. Protoplasts can also be used as recipients for isolated DNA, again in the presence of polyethylene glycol (PEG). In our study we had undertaken random genome shuffling by protoplast fusion of two, rather poorly expressed actinomycetes strains A (Figure 1) & B (Figure 2), mediated by PEG; and also by naked DNA transformation of Strain A protoplast with the DNA of Strain B. We generated eight protoplast fusants and seven transformants from parents considering their morphological difference from the two parent strains. These 15 recombinants were checked for their same colony morphologies for five generations to ensure phenotypic stability. Antibiotic resistance pattern was established by using antibiotic octodisc to generate a marker profile of the recombinants and the parent strains. Eight fusants (AP-18, AP-25, AP-2, AP-11, AP-14, AP-19, AP-11 and AP-27) and four transformants (TAP-30, TAP-31, TAP-32 and TAP-33) (Table 1) have shown a different antibiotic sensitivity pattern as compared to the parent strains. We envisage that these recombinants harbor shuffled gene clusters. To support array of conditions to express such shuffled/cryptic genes the recombinants were fermented in 11 different nutrient stress variants. The extracts generated were subjected to metabolite profiling by HPLC-ELSD, bioactivity screening for cytotoxicity and anti-infective capabilities. Two fusants AP-11 (Figure 3) and AP-25; one transformant TAP-32 (in growth media MBA-5 and MBA-7) displayed antifungal activity unlike parent strains (Table 2) Fusant AP-11 (Table 5) exhibited significant cell growth inhibition of five different cancer cell lines. The parents Strain A and Strain B did not exhibit any cell growth inhibition of these cell lines (Table 5). The metabolite profiling of fusant AP-11 and transformant TAP-32 was done by HPLC-ELSD. AP-11 showed the presence of five additional peaks (Figure 5 & Figure 6); TAP-32 extract from medium MBA-5 (Figure 7 & Figure 8) showed the presence of four additional peaks and TAP-32 extract from MBA-7 (Figure 9 & Figure 10) showed 14 additional peaks as compared to parent strains in similar medium and media controls. The study indicated that protoplast fusion and transformation have not only caused morphological changes but also shuffled genes responsible for synthesis of bioactive molecules. Further characterization of these new peaks is warranted.
Shrikant Anant, Ph.D.
The Department of Molecular & Integrative Physiology, Kansas University Medical Center, USA
Cancer Stem Cells: Target Colon Cancers
Shrikant Anant, Deep Kwatra and Dharmalingam Subramaniam
Colon cancer is a leading cause of cancer related deaths in the US, and its rate is increasing at an alarming rate in lndia. Recent studies have suggested the drug resistance role for a mall number of cells within a tumor called cancer stem cells. We identified the colon cancer stem cell marker DCLK1, a member of the protein kinase superfamily and the doublecortin family. The protein encodes a Cterminal serinethreonine protein kinase domain, which shows substantial homology to Ca2calmodulindependent protein kinase. Our current studies have been to identify compounds that can either affect DCLK1 expression or inhibits its activity as a way to inhibit cancer stem cells. Honokiol is a biphenolic compound that has been used in the traditional Chinese Medicine for treating various ailments. In vitro kinase assays with recombinant DCLK1 demonstrated that honokiol inhibits its kinase activity in a dose dependent manner. We therefore determined the effect of honokiol on stem cells. One method to look at effects on stem cells is perform a spheroid assay, where spheroids formation is suggested to maintain stemlike characteristic of cancer cells. Honokiol significantly suppressed colonosphere formation of two colon cancer cell lines HCT116 and SW480. Flow cytometry studies confirmed that honokiol reduced the number of DCLK1cells. A critical signaling pathway known to modulate intestinal stem cell proliferation is the Hippo signaling pathway, and deregulation of the pathway leads to tumor development. DCLK1cells had high levels of YAP1, the nuclear target of Hippo signaling. We determined the effect of honokiol on components of the hipposignaling pathway. Honokiol reduced the phosphorylation of Mst1/2, Lats1/2 and YAP1. Furthermore, honokiol treatment resulted in downregulation of YAPTEAD complex protein TEAD-1. Ectopic expression of the TEAD-1 partially rescued the cells from honokiol mediated growth suppression. To determine the effect of honokiol on tumor growth in vivo, nude mice harboring HCT116 tumor xenografts in their flanks were administered the compound intraperitoneally every day for 21 days. Honokiol treatment significantly inhibited tumor xenograft growth. Western blot and immunohistochemistry analyses demonstrated significant inhibition in the expression of stem marker and Hippo signaling proteins in the honokioltreated xenograft tissues. Taken together, these data suggest that honokiol is a potent inhibitor of colon cancer that targets DCLK1 stem cells by inhibiting Hippo signaling pathway.
Sunilkumar Sukumaran, Ayyappan Nair, Madhuri Subbiah, Gunja Gupta, Lakshmi Rajakrishna, Pradeep Savanoor Raghavendra, Subbulakshmi Karthikeyan, Salini Krishnan Unni and Ganesh Sambasivam
Genotoxicity is defined as DNA damage that leads to gene mutations which can become tumorigenic. Genotoxicity testing is important to ensure drug safety and is mandatory prior to Phase I/II clinical trials of new drugs. The results from genetic toxicology studies help to identify hazardous drugs and environmental genotoxins. Currently, among others there are four tests recommended by regulatory authorities (Ames test-bacterial, chromosome aberrations; in vitro gene mutation-eukaryotic cells and in vivo test). These assays are laborious, time consuming, require large quantities of test compounds and limited by throughput challenges. The site and mechanism of genotoxicity are not revealed by these assays and data obtained from bacterial tests might not translate the same in mammals. To address these we have developed a novel, versatile, human cell based, high throughput, reporter based genotoxicity screen (Anthem’s Genotox screen). This screen is performed on genetically engineered human cell lines that express 3 reporter genes under transcriptional control of ‘early DNA damage sensors’ (p53, p21 and GADD153). These genes are involved in DNA repair, cell cycle arrest and/or apoptosis. p21 and GADD are also known to be induced in a p53 independent manner. p53 blocks G1/S transition of cell cycle while the p53 independent DNA damage block G2/M transition. Identification of the mechanism of genotoxicity helps in rational drug designing. Additionally, the platform can be used to screen other potential genotoxins from cosmetics, food and environment. Initial validation studies of the Genotox screen was performed with over 60 compounds chosen from a variety of chemical classes. The genotoxic potential of metabolites was tested using rat liver S9 fractions. The results demonstrated a sensitivity of 86.7–92.3% and a specificity of 70–78.6% when compared with currently available in vitro genotoxicity assays. This Genotox screen would prove to be an invaluable human cell based tool to weed out potential genotoxins in various industries.