Butyrate and propionate downregulate ERK phosphorylation in HT-29 colon carcinoma cells prior to differentiation.

We have characterized the effects of different short-chain fatty acids (SCFAs) on cell growth and differentiation as well as the phosphorylation state of ERK1 and 2 in the human colon adenocarcinoma cell line HT-29. Of the five SCFAs tested, only butyrate and propionate impaired cellular proliferation. Moreover, butyrate and propionate specifically resulted in a decrease in ERK1 and 2 phosphorylation at 3 and 6 hours post-treatment, suggesting a correlation between the ability of these SCFAs to inhibit cellular proliferation and decrease ERK phosphorylation. Notably, the decrease in ERK phosphorylation was observed prior to the induction of the differentiation markers alkaline phosphatase (AP) and carcinoembryonic antigen (CEA) by butyrate and propionate from days 6 to 18 post-treatment. In the case of butyrate- and propionate-induced differentiation, ERK phosphorylation is a marker and may play a role in the proliferation and/or differentiation states of this cell line.

ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells.

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 microM. Likewise, Western blot analysis demonstrated that addition of 3 microM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.

Analysis of the basal and inducible activities of the ICPO promoter of herpes simplex virus type 1.

Sequences from -420 to -70 from the ICPO transcriptional start site of herpes simplex virus type 1 are dispensable for reactivation from latency. A putative cAMP-response element (CRE) outside of this region was non-functional in both murine neuroblastoma (NB41A3) and rat pheochromocytoma (PC12) cells. Also, poor binding of cAMP-response element binding protein (CREB) was observed. Sequences from -95 to -37 are important for constitutive activity in NB41A3, PC12 and baby hamster kidney (BHK) cells. The TATA box and Sp1 site were also shown to be major contributors to constitutive activity. Finally, high constitutive activity of a deleted construct (-420 to -1) in NB41A3 and BHK cells suggests transcription initiates upstream of -420 in the absence of VP16. The implications of these observations regarding ICPO expression during the virus life-cycle are discussed.

Role of cis-acting sequences of the ICPO promoter of herpes simplex virus type 1 in viral pathogenesis, latency and reactivation.

A mutant herpes simplex virus type 1, termed delta Tfi, with a 350 bp deletion of the Sp1, NF-kappaB, TAATGARATs, C/EBP and F2 DNA-binding elements from -420 to -70 relative to the transcriptional start site of ICPO (Vmw 110), was generated and characterized. The efficiency of plating of delta Tfi was reduced on Vero cells and it expressed correctly initiated ICPO RNA in the presence of cycloheximide, although RNA levels were 2.5-fold lower than with wild-type (KOS) and marker-rescued (delta TfiR) viruses. This was consistent with a demonstrated reduction in ICPO protein expression for delta Tfi at early times post-infection and a 3-fold reduction in ICPO-dependent transactivation of the ICP6 promoter. KOS, delta Tfi and delta TfiR replication in murine corneas and trigeminal ganglia were comparable when measured on a complementing cell line, but delta Tfi titres appeared 15- to 50-fold lower when measured on Vero cells. delta Tfi was correspondingly less virulent than wild-type or marker-rescued viruses in both immunocompetent and SCID mice. delta Tfi, however, established and reactivated from latency with efficiencies comparable to wild-type and marker-rescued viruses. These results demonstrate that although this deletion of the ICPO promoter results in lower levels of ICPO in vitro and decreased virulence in vivo, the establishment of and reactivation from latency are unaffected. This indicates that elements which regulate ICPO expression and virulence during acute infection may be distinct from those involved in reactivation.

The roles of the cAMP-response element and TATA box in expression of the herpes simplex virus type 1 latency-associated transcripts.

A quantitative ribonuclease protection assay (RPA) was developed in order to rapidly and accurately measure the levels and timing of latency-associated transcript (LAT) expression in ganglia latently infected with wild-type and mutant herpes simplex virus (HSV). Use of this assay in parallel with measurement of viral titers in murine trigeminal ganglia demonstrated that the peak of viral replication precedes the peak and subsequent plateau of LAT expression. This plateau of LAT expression was unaltered from Day 7 through the end of the experimental period on Day 28, suggesting that LAT does not further accumulate during latency of wild-type virus. RPA analyses of trigeminal ganglia latently infected with HSV-1 mutants containing specific alterations in the LAT TATA box, cyclic AMP-response element (CRE), and both TATA and CRE were performed. Mutation of the upstream TATA box reduced LAT expression to 25% of wild-type or marker-rescued virus levels, whereas mutation of the CRE did not significantly affect LAT expression in vivo whether in the presence or absence of the TATA box. These experiments demonstrate a specific requirement for the upstream promoter TATA box for wild-type LAT expression. Further examination of the role of the CRE and the TATA box by transient expression assays suggests that the CRE is important for inducible activity and that its interaction with the TATA box requires stereospecific alignment.