The bioimpedance spectroscopy (BIS) technique is potentially a useful tool to differentiate malignancy based on the variation of electrical properties presented by different tissues and cells. The different tissues and cells present variant electrical resistance and reactance when excited at different frequencies. The main purpose of this area of research is to use impedance measurements over a low-frequency bandwidth ranging from 1 kHz to 3 MHz to 1) differentiate the pathological stages of cancer cells under laboratory conditions and 2) permit the extraction of electrical parameters related to cellular information for further analysis. This provides evidence to form the basis of bioimpedance measurement at the cellular level and aids the potential future development of rapid diagnostics from biopsy materials. Three cell lines, representing normal breast epithelia and different pathological stages of breast cancer, have been measured using a standard impedance analyzer driving a four-electrode chamber filled with different cell suspensions. We identify the specific BIS profile for each cell type and determine whether these can be differentiated. In addition, the electrical parameters, e.g., the intracellular conductivity, membrane capacitance/capacity, characteristic frequency, are extracted by the use of equivalent circuit models and physical models to provide details of the cell electric signatures for further analysis of cancer cell pathology.

To explore the role of p16^INK4a as an intrinsic barrier to B cell transformation by EBV, we transformed primary B cells from an individual homozygous for a deletion in the CDKN2A locus encoding p16^INK4a and p14^ARF. Using recombinant EBV-BAC viruses expressing conditional EBNA3C (3CHT), we developed a system that allows inactivation of EBNA3C in lymphoblastoid cell lines (LCLs) lacking active p16^INK4a protein but expressing a functional 14^ARF-fusion protein (p14/p16). The INK4a locus is epigenetically repressed by EBNA3C – in cooperation with EBNA3A – despite the absence of functional p16^INK4a. Although inactivation of EBNA3C in LCLs from normal B cells leads to an increase in p16^INK4a and growth arrest, EBNA3C inactivation in the p16^INK4a-null LCLs has no impact on the rate of proliferation, establishing that the repression of INK4a is a major function of EBNA3C in EBV-driven LCL proliferation. This conditional LCL system allowed us to use microarray analysis to identify and confirm genes regulated specifically by EBNA3C, independently of proliferation changes modulated by the p16^INK4a-Rb-E2F axis. Infections of normal primary B cells with recombinant EBV-BAC virus from which EBNA3C is deleted or with 3CHT EBV in the absence of activating ligand 4-hydroxytamoxifen, revealed that EBNA3C is necessary to overcome an EBV-driven increase in p16^INK4a expression and concomitant block to proliferation 2–4 weeks post-infection. If cells are p16^INK4a-null, functional EBNA3C is dispensable for the outgrowth of LCLs. Epstein-Barr virus (EBV) is a causative agent of several types of B cell lymphoma. In human B cells, EBV reduces protein levels of at least two tumour suppressors that would otherwise be activated in response to over-expressed oncogenes. These proteins are BIM, which induces cell death and p16^INK4a, which prevents cell proliferation. Repression of both is via epigenetic methylation of histones and is dependent on expression of both EBNA3A and EBNA3C – two EBV proteins required for the transformation of normal B cells into lymphoblastoid cell lines (LCLs). In this report we have used EBV with a conditionally active EBNA3C – active only in the presence of 4-hydroxytamoxifen – together with B cells from an individual carrying a homozygous deletion of p16^INK4a to confirm that regulation of p16^INK4a expression is a major function of EBNA3C and demonstrate that if B cells lack p16^INK4a, then EBNA3C is no longer required for EBV-driven proliferation of LCLs. Furthermore we show that early after the infection of normal B cells, EBV induces p16^INK4a accumulation that – if unchecked by EBNA3C (and EBNA3A) – prevents LCL outgrowth. Formal proof that p16^INK4a is the main target of EBNA3C comes with the production of p16-null LCLs that have never expressed functional EBNA3C.

The human gammaherpesviruses Epstein–Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) both infect lymphoid and epithelial cells and both are implicated in the development of cancer. The two viruses establish latency in B-lymphoid cells that, once disrupted, leads to a burst of virus replication during the lytic cycle. A basic leucine zipper (bZIP) transcription factor encoded by EBV, Zta (also known as BZLF1 and ZEBRA), is key to the disruption of EBV latency. KSHV encodes a related protein, K-bZIP (also known as RAP and K8α). Recent developments in our understanding of the structures and functions of these two viral bZIP proteins have led to the conclusion that they are not homologues. Two important features of Zta are its ability to interact directly with DNA and to induce EBV replication whereas K-bZIP is not known to interact directly with DNA or to induce KSHV replication. Despite these differences, the ability to disrupt cell cycle control is conserved in both Zta and K-bZIP. The interactions of Zta and K-bZIP with cellular genes will be reviewed here.

Thyroid-specific expression of the rat thyroglobulin gene is mediated by transcriptional control. Sufficient DNA sequence information to confer thyroid-specific expression to a heterologous gene is contained between positions −168 and +39. DNA-binding studies have demonstrated that this region interacts with two thyroid-specific factors (TTF-1 and TTF-2), and a ubiquitous factor (UFA). Here we have characterized three elements within the promoter, A, K, and C, which are important for promoter activity in thyroid cells. We have shown by mutational analysis that the interaction of TTF-1 with the A and C regions, UFA with the A region, and TTF-2 with the K region are required for full promoter activity. The complex interactions in the A region can be replaced by the substitution of the UFA/TTF-1-binding site with a high-affinity TTF-1 binding site. There is a correlation between the presence of TTF-1 and TTF-2 DNA-binding activities and the expression of thyroglobulin, which implies that the mechanism restricting thyroglobulin expression to thyroid cells is mediated through the control of the expression, or the activity, of TTF-1 and TTF-2.

The ability of Epstein-Barr virus (EBV) to establish latency allows it to evade the immune system and to persist for the lifetime of its host; one distinguishing characteristic is the lack of transcription of the majority of viral genes. Entry into the lytic cycle is coordinated by the viral transcription factor, Zta (BZLF1, ZEBRA, and EB1), and downstream effectors, while viral genome replication requires the concerted action of Zta and six other viral proteins at the origins of lytic replication. We explored the chromatin context at key EBV lytic cycle promoters (BZLF1,BRLF1,BMRF1, andBALF5) and the origins of lytic replication during latency and lytic replication. We show that a repressive heterochromatin-like environment (trimethylation of histone H3 at lysine 9 [H3K9me3] and lysine 27 [H3K27me3]), which blocks the interaction of some transcription factors with DNA, encompasses the key early lytic regulatory regions. Epigenetic silencing of the EBV genome is also imposed by DNA methylation during latency. The chromatin environment changes during the lytic cycle with activation of histones H3, H4, and H2AX occurring at both the origins of replication and at the key lytic regulatory elements. We propose that Zta is able to reverse the effects of latency-associated repressive chromatin at EBV early lytic promoters by interacting with Zta response elements within the H3K9me3-associated chromatin and demonstrate that these interactions occurin vivo. Since the interaction of Zta with DNA is not inhibited by DNA methylation, it is clear that Zta uses two routes to overcome epigenetic silencing of its genome.