Clinically relevant humanized mouse models of metastatic prostate cancer facilitate therapeutic evaluation.

There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate is anatomically and developmentally different from the human prostate and does not spontaneously form tumors. Genetically engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts are an alternative but must rely on an immunocompromised host. Therefore, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of androgen receptor-targeted and immunotherapies. These mice maintain multiple human immune cell lineages, including functional human T-cells and myeloid cells. Implications: To our knowledge, results illustrate the first model of human PCa that has an intact human immune system, metastasizes to clinically relevant locations, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.

Clinically relevant humanized mouse models of metastatic prostate cancer to evaluate cancer therapies.

There is tremendous need for improved prostate cancer (PCa) models. The mouse prostate does not spontaneously form tumors and is anatomically and developmentally different from the human prostate. Engineered mouse models lack the heterogeneity of human cancer and rarely establish metastatic growth. Human xenografts represent an alternative but rely on an immunocompromised host. Accordingly, we generated PCa murine xenograft models with an intact human immune system (huNOG and huNOG-EXL mice) to test whether humanizing tumor-immune interactions would improve modeling of metastatic PCa and the impact of hormonal and immunotherapies. These mice maintain multiple human cell lineages, including functional human T-cells and myeloid cells. In 22Rv1 xenografts, subcutaneous tumor size was not significantly altered across conditions; however, metastasis to secondary sites differed in castrate huNOG vs background-matched immunocompromised mice treated with enzalutamide (enza). VCaP xenograft tumors showed decreases in growth with enza and anti-Programed-Death-1 treatments in huNOG mice, and no effect was seen with treatment in NOG mice. Enza responses in huNOG and NOG mice were distinct and associated with increased T-cells within tumors of enza treated huNOG mice, and increased T-cell activation. In huNOG-EXL mice, which support human myeloid development, there was a strong population of immunosuppressive regulatory T-cells and Myeloid-Derived-Suppressor-Cells (MDSCs), and enza treatment showed no difference in metastasis. Results illustrate, to our knowledge, the first model of human PCa that metastasizes to clinically relevant locations, has an intact human immune system, responds appropriately to standard-of-care hormonal therapies, and can model both an immunosuppressive and checkpoint-inhibition responsive immune microenvironment.

The wheat chloroplastic small heat shock protein (sHSP26) is involved in seed maturation and germination and imparts tolerance to heat stress.

The nuclear-encoded chloroplast small heat shock proteins (sHSPs) are present in all plant species from algae to angiosperms. Expression analysis shows that the wheat chloroplastic sHSP (HSP26) is highly inducible by heat stress in almost all the vegetative and generative tissues and is also expressed constitutively in certain developmental growth stages. We characterize wheat chloroplastic sHSP 26 through transgenic approach using Arabidopsis and report cloning of the promoter and its characterization. Transgenic Arabidopsis plants were substantially tolerant under continuous high temperature regimen than wild-type plants, as measured by photosystem II (PSII) activity, accumulation of more photosynthetic pigments, higher biomass and seed yield. Transgenic plants produced bold seeds under high temperature, having higher germination potential than the wild-type plants. Further, antisense Arabidopsis plants showed negligible tolerance even for non-lethal heat shock, impaired in basal thermo-tolerance, and accumulated less biomass and seed yield under normal growth conditions. Promoter analysis revealed the presence of several heat and other abiotic stress responsive cis-acting elements along with developmental stage and tissue-specific elements. Analysis of promoter through GUS reporter system in both transgenic rice and Arabidopsis further confirms the role of chloroplastic sHsp26 in heat and other abiotic stresses as well as during seed maturation and germination. Genome-wide expression analysis of overexpression Arabidopsis plants revealed that the transcriptome remained unchanged in the transgenic plants and the tolerance was due to the overexpression of chloroplastic heat shock protein (HSP) only.

Expression analysis of a heat-inducible, Myo-inositol-1-phosphate synthase (MIPS) gene from wheat and the alternatively spliced variants of rice and Arabidopsis.

Molecular dissection and a deeper analysis of the heat stress response mechanism in wheat have been poorly understood so far. This study delves into the molecular basis of action of TaMIPS, a heat stress-inducible enzyme that was identified through PCR-select subtraction technology, which is named here as TaMIPS2. MIPS (L-Myo-inositol-phosphate synthase) is important for the normal growth and development in plants. Expression profiling showed that TaMIPS2 is expressed during different developing seed stages upon heat stress. Also, the transcript levels increase in unfertilized ovaries and significant amounts are present during the recovery period providing evidence that MIPS is crucial for its role in heat stress recovery and flower development. Alternatively spliced forms from rice and Arabidopsis were also identified and their expression analysis revealed that apart from heat stress, some of the spliced variants were also inducible by drought, NaCl, Cold, ABA, BR, SA and mannitol. In silico promoter analysis revealed various cis-elements that could contribute for the differential regulation of MIPS in different plant systems. Phylogenetic analysis indicated that MIPS are highly conserved among monocots and dicots and TaMIPS2 grouped specifically with monocots. Comparative analyses was undertaken by different experimental approaches, i.e., semi-quantitative RT-PCR, quantitative RT-PCR, Genevestigator as a reference expression tool and motif analysis to predict the possible function of TaMIPS2 in regulating the different aspects of plant development under abiotic stress in wheat.

Identification and characterization of high temperature stress responsive genes in bread wheat (Triticum aestivum L.) and their regulation at various stages of development.

To elucidate the effect of high temperature, wheat plants (Triticum aestivum cv. CPAN 1676) were given heat shock at 37 and 42°C for 2 h, and responsive genes were identified through PCR-Select Subtraction technology. Four subtractive cDNA libraries, including three forward and one reverse subtraction, were constructed from three different developmental stages. A total of 5,500 ESTs were generated and 3,516 high quality ESTs submitted to Genbank. More than one-third of the ESTs generated fall in unknown/no hit category upon homology search through BLAST analysis. Differential expression was confirmed by cDNA macroarray and by northern/RT-PCR analysis. Expression analysis of wheat plants subjected to high temperature stress, after 1 and 4 days of recovery, showed fast recovery in seedling tissue. However, even after 4 days, recovery was negligible in the developing seed tissue after 2 h of heat stress. Ten selected genes were analyzed in further detail including one unknown protein and a new heat shock factor, by quantitative real-time PCR in an array of 35 different wheat tissues representing major developmental stages as well as different abiotic stresses. Tissue specificity was examined along with cross talk with other abiotic stresses and putative signalling molecules.

Heat shock factors in rice (Oryza sativa L.): genome-wide expression analysis during reproductive development and abiotic stress.

Plants respond to heat stress by enhancing the expression of genes encoding heat shock protein (HSPs) genes through activation of heat shock factors (HSFs) which interact with heat shock elements present in the promoter of HSP genes. Plant HSFs have been divided into three conserved classes viz A, B and C. In the present study, a detailed analysis has been done of all rice HSFs, along with their spliced variants. Their chromosomal localization reveals that six HSFs are segmentally duplicated and four pairs of these segmentally duplicated HSF encoding genes show pseudo-functionalization. Expression profiling through microarray and quantitative real-time PCR showed that eight OsHsfs express at a higher level during seed development, while six HSFs are up-regulated in all the abiotic stresses studied. The expression of OsHsfA2a gene in particular was greatly stimulated by heat stress in both root and shoot tissues and also during panicle and seed development. OsHsfA3 was found more responsive to cold and drought stress, while OsHsfA7 and OsHsfA9 showed developing seed-specific expression. This study also revealed that spliced variants generally accumulated at a higher level in all the tissues examined. Different hormones/elicitors like ABA, brassinosteroids and salicylic acid also alter OsHsf gene expression. Calcium in combination with heat stress elevated further the level of HSF transcripts. Expression analysis by both microarray and real-time RT-PCR revealed a unique stable constitutive expression of OsHsfA1 across all the tissues and stresses. A detailed in silico analysis involving identification of unidentified domains has been done by MEME-motif tool in their full-length proteins as well as in DNA-binding domains. Analysis of 1 kb putative promoter region revealed presence of tissue-specific, abiotic stress and hormone-related cis-acting elements, correlating with expression under stress conditions.

Wheat chloroplast targeted sHSP26 promoter confers heat and abiotic stress inducible expression in transgenic Arabidopsis Plants.

The small heat shock proteins (sHSPs) have been found to play a critical role in physiological stress conditions in protecting proteins from irreversible aggregation. To characterize the hloroplast targeted sHSP26 promoter in detail, deletion analysis of the promoter is carried out and analysed via transgenics in Arabidopsis. In the present study, complete assessment of the importance of CCAAT-box elements along with Heat shock elements (HSEs) in the promoter of sHSP26 was performed. Moreover, the importance of 5' untranslated region (UTR) has also been established in the promoter via Arabidopsis transgenics. An intense GUS expression was observed after heat stress in the transgenics harbouring a full-length promoter, confirming the heat-stress inducibility of the promoter. Transgenic plants without UTR showed reduced GUS expression when compared to transgenic plants with UTR as was confirmed at the RNA and protein levels by qRT-PCR and GUS histochemical assays, thus suggesting the possible involvement of some regulatory elements present in the UTR in heat-stress inducibility of the promoter. Promoter activity was also checked under different abiotic stresses and revealed differential expression in different deletion constructs. Promoter analysis based on histochemical assay, real-time qPCR and fluorimetric analysis revealed that HSEs alone could not transcribe GUS gene significantly in sHSP26 promoter and CCAAT box elements contribute synergistically to the transcription. Our results also provide insight into the importance of 5`UTR of sHsp26 promoter thus emphasizing the probable role of imperfect CCAAT-box element or some novel cis-element with respect to heat stress.

A seed preferential heat shock transcription factor from wheat provides abiotic stress tolerance and yield enhancement in transgenic Arabidopsis under heat stress environment.

Reduction in crop yield and quality due to various abiotic stresses is a worldwide phenomenon. In the present investigation, a heat shock factor (HSF) gene expressing preferentially in developing seed tissues of wheat grown under high temperatures was cloned. This newly identified heat shock factor possesses the characteristic domains of class A type plant HSFs and shows high similarity to rice OsHsfA2d, hence named as TaHsfA2d. The transcription factor activity of TaHsfA2d was confirmed through transactivation assay in yeast. Transgenic Arabidopsis plants overexpressing TaHsfA2d not only possess higher tolerance towards high temperature but also showed considerable tolerance to salinity and drought stresses, they also showed higher yield and biomass accumulation under constant heat stress conditions. Analysis of putative target genes of AtHSFA2 through quantitative RT-PCR showed higher and constitutive expression of several abiotic stress responsive genes in transgenic Arabidopsis plants over-expressing TaHsfA2d. Under stress conditions, TaHsfA2d can also functionally complement the T-DNA insertion mutants of AtHsfA2, although partially. These observations suggest that TaHsfA2d may be useful in molecular breeding of crop plants, especially wheat, to improve yield under abiotic stress conditions.