Vitamin D and docosahexaenoic acid inhibit proliferation of the ovarian cancer cell line OVCAR4.

BACKGROUND: Ovarian cancer is one of the deadliest cancers in women. Improved preventative, diagnostic, and therapeutic strategies are needed. Certain dietary patterns and nutrients such as vitamin D and omega-3 fatty acids are associated with reduced cancer risk, but their effects on ovarian cancer remain to be fully elucidated, and their combined effects have not been explored. AIM: To determine the individual and combined effects of the active vitamin D metabolite, calcitriol, and the omega-3 fatty acid, docosahexaenoic acid, on cell growth, and the abundance of the vitamin D receptor (VDR), proteins that modulate cell cycle progression, and apoptotic markers. METHODS: OVCAR4 cells, a model of ovarian cancer, were treated with calcitriol, and docosahexaenoic acid, either alone or in combination. Effects on cell growth were determined by the sulforhodamine B assay. Changes in VDR, the cell cycle promotor c-Myc, the cell cycle inhibitor p27 and cleaved PARP, were determined by Western blotting. RESULTS: While OVCAR4 cell growth was inhibited by individual treatment with either calcitriol or docosahexaenoic acid, the combined treatment revealed enhanced growth inhibition as compared to either treatment alone. Furthermore, long-term treatment (12 days) yielded stronger growth inhibition at lower concentrations as compared to short-term treatments (3 days). Accompanying this growth inhibition was a decrease in c-Myc, and an increase in p27. CONCLUSIONS: The observed reduction in cell growth mediated by calcitriol and docosahexaenoic acid highlights the need for further research utilizing these nutrients, alone and especially in combination, to support ovarian cancer prevention and treatment.

Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation.

BACKGROUND: Cell division is positively regulated by cyclin-dependent kinases (CDKs) partnered with cyclins and negatively regulated by CDK inhibitors. In the frog, Xenopus laevis, three types of CDK inhibitors have been described: p27Xic1 (Xic1) which shares sequence homology with both p21Cip1 and p27Kip1 from mammals, p16Xic2 (Xic2) which shares sequence homology with p21Cip1, and p17Xic3 (Xic3) which shares sequence homology with p27Kip1. While past studies have demonstrated that during DNA polymerase switching, Xic1 is targeted for protein turnover dependent upon DNA, Proliferating Cell Nuclear Antigen (PCNA), and the ubiquitin ligase CRL4Cdt2, little is known about the processes that regulate Xic2 or Xic3. METHODS: We used the Xenopus interphase egg extract as a model system to examine the regulation of Xic2 by proteolysis and phosphorylation. RESULTS: Our studies indicated that following primer synthesis during the initiation of DNA replication, Xic2 is targeted for DNA- and PCNA-dependent ubiquitin-mediated proteolysis and that Cdt2 can promote Xic2 turnover. Additionally, during interphase, Xic2 is phosphorylated by CDK2 at Ser-98 and Ser-131 in a DNA-independent manner, inhibiting Xic2 turnover. In the presence of double-stranded DNA ends, Xic2 is also phosphorylated at Ser-78 and Ser-81 by a caffeine-sensitive kinase, but this phosphorylation does not alter Xic2 turnover. Conversely, in the presence or absence of DNA, Xic3 was stable in the Xenopus interphase egg extract and did not exhibit a shift indicative of phosphorylation. CONCLUSIONS: During interphase, Xic2 is targeted for DNA- and PCNA-dependent proteolysis that is negatively regulated by CDK2 phosphorylation. During a response to DNA damage, Xic2 may be alternatively regulated by phosphorylation by a caffeine-sensitive kinase. Our studies suggest that the three types of Xenopus CDK inhibitors, Xic1, Xic2, and Xic3 appear to be uniquely regulated which may reflect their specialized roles during cell division or early development in the frog.

Microarray-based analysis of cell-cycle gene expression during spermatogenesis in the mouse.

Mammalian spermatogenesis is a continuum of cellular differentiation in a lineage that features three principal stages: 1) a mitotically active stage in spermatogonia, 2) a meiotic stage in spermatocytes, and 3) a postreplicative stage in spermatids. We used a microarray-based approach to identify changes in expression of cell-cycle genes that distinguish 1) mitotic type A spermatogonia from meiotic pachytene spermatocytes and 2) pachytene spermatocytes from postreplicative round spermatids. We detected expression of 550 genes related to cell-cycle function in one or more of these cell types. Although a majority of these genes were expressed during all three stages of spermatogenesis, we observed dramatic changes in levels of individual transcripts between mitotic spermatogonia and meiotic spermatocytes and between meiotic spermatocytes and postreplicative spermatids. Our results suggest that distinct cell-cycle gene regulatory networks or subnetworks are associated with each phase of the cell cycle in each spermatogenic cell type. In addition, we observed expression of different members of certain cell-cycle gene families in each of the three spermatogenic cell types investigated. Finally, we report expression of 221 cell-cycle genes that have not previously been annotated as part of the cell cycle network expressed during spermatogenesis, including eight novel genes that appear to be testis-specific.

Non-catalytic function for ATR in the checkpoint response.

The ATR family of checkpoint kinases is essential for an appropriate response to genomic insults in eukaryotes. Included in this family are Mei-41 in Drosophila, Mec1 inS. cerevisiae, Rad3 in S. pombe, and ATR in vertebrates. These large kinases phosphorylateand modify multiple cell cycle and checkpoint factors, leading to cell cycle arrest, DNA repair, and induction of apoptosis. The catalytic domain of all ATR family members comprises only a fraction of the total protein. Here, we show that the non-catalytic portion of ATR has a conserved function in the checkpoint response. Expression of either wild type or various kinase defective forms of Xenopus ATR (XATR) in S. cerevisiae mec1 mutants suppresses the checkpoint defect and induces a DNA damage dependent mitotic cell cycle arrest. This suppression requires the presence of yeast Ddc2 and Rad9 but functions independently of Rad9 modification and Rad53 activation. Our results indicate that XATR is not functioning through the established mitotic checkpoint pathways. Instead, we find that the XATR suppression of the mec1 mutant checkpoint defect requires the spindle checkpoint factors Mad1 and Mad2, suggesting a role for XATR in the spindle assembly checkpoint. Finally, we show that a yeast strain expressing a truncated, kinase domain deleted form of mec1 from the endogenous locus is partially checkpoint proficient and induces a mitotic cell cycle arrest in a Mad2 dependent manner. Thus, the link between the non-catalytic region of the ATR kinase family and the spindle checkpoint pathway is conserved.

Measurement of Wee kinase activity.

The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activity of the Wee kinases are regulated during the cell cycle and development. In this chapter, we describe several procedures to measure the activity of the Wee kinases found either in crude extracts or in purified preparations. Specific protocols include the production and purification of recombinant Cdc2/Cyclin B substrate, the production of crude subcellular extract fractions, the purification of endogenous or recombinant Wee kinases, Wee kinase assays, and the Histone H1 kinase assay to measure Cdc2 activity. In addition, support protocols are provided that describe the use and production of Ni-IDA beads for the purification of Histidine-tagged proteins, and the use of the baculovirus expression system to produce recombinant proteins.

Xenopus Cds1 is regulated by DNA-dependent protein kinase and ATR during the cell cycle checkpoint response to double-stranded DNA ends.

The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus Cds1 (XCds1). Under normal cell cycle conditions, nonactivated XCds1 constitutively associates with a Xenopus ATR complex. The association of XCds1 with this complex does not require a functional forkhead activation domain but does require a putative SH3 binding region that is found in XCds1. In response to double-stranded DNA ends, the amino terminus of XCds1 is rapidly phosphorylated in a sequential pattern. First DNA-PK phosphorylates serine 39, a site not previously recognized as important in Cds1 regulation. Xenopus ATM, ATR, and/or DNA-PK then phosphorylate three consensus serine/glutamine sites. Together, these phosphorylations have the dual function of inducing dissociation from the ATR complex and independently promoting the full activation of XCds1. Thus, the checkpoint-mediated activation of XCds1 requires phosphorylation by multiple phosphoinositide 3-kinase-related kinases, protein-protein dissociation, and autophosphorylation.

Inhibition of the cell cycle is required for convergent extension of the paraxial mesoderm during Xenopus neurulation.

Coordination of morphogenesis and cell proliferation is essential during development. In Xenopus, cell divisions are rapid and synchronous early in development but then slow and become spatially restricted during gastrulation and neurulation. One tissue that transiently stops dividing is the paraxial mesoderm, a dynamically mobile tissue that forms the somites and body musculature of the embryo. We have found that cessation of cell proliferation is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopus embryo. Instrumental in this cell cycle arrest is Wee2, a Cdk inhibitory kinase that is expressed in the paraxial mesoderm from mid-gastrula stages onwards. Morpholino-mediated depletion of Wee2 increases the mitotic index of the paraxial mesoderm and this results in the failure of convergent extension and somitogenesis in this tissue. Similar defects are observed if the cell cycle is inappropriately advanced by other mechanisms. Thus, the low mitotic index of the paraxial mesoderm plays an essential function in the integrated cell movements and patterning of this tissue.

Multiple Cdk1 inhibitory kinases regulate the cell cycle during development.

The Wee kinases block entry into mitosis by phosphorylating and inhibiting the activity of the mitotic cyclin-dependent kinase, Cdk1. We have found that the various Xenopus Wee kinases have unique temporal and spatial patterns of expression during development. In addition, we have isolated and characterized a new Wee1-like kinase, Xenopus Wee2. By both in vivo and in vitro tests, Xenopus Wee2 functions as a Wee1-like kinase. The previously isolated Wee1-like kinase, Xenopus Wee1, is expressed only as maternal gene product. In contrast, Xenopus Wee2 is predominantly a zygotic gene product, while the third Wee kinase, Xenopus Myt1, is both a maternal and zygotic gene product. Concurrent with the changing levels of these Cdk inhibitory kinases, the pattern of embryonic cell division becomes asynchronous and spatially restricted in the Xenopus embryo. Interestingly, once zygotic transcription begins, Xenopus Wee2 is expressed in regions of the embryo that are devoid of mitotic cells, such as the involuting mesoderm. In contrast, Xenopus Myt1 is expressed in regions of the embryo that have high levels of proliferation, such as the developing neural tissues. The existence of multiple Wee kinases may help explain how distinct patterns of cell division arise and are regulated during development.