Noninvasive Monitoring of Radiation-Induced Salivary Gland Vascular Injury.

Xerostomia is a common side effect of radiation therapy (RT) in patients with head and neck cancer. However, limited information is available on the temporal dynamics of parenchymal and vascular changes in salivary glands following RT. To address this gap in knowledge, we conducted experimental studies in mice employing ultrasound (US) with coregistered photoacoustic imaging (PAI) to noninvasively assess the early and late changes in salivary gland size, structure, vascularity, and oxygenation dynamics following RT. Multiparametric US-PAI of salivary glands was performed in immune-deficient and immune-competent mice before and after RT along with correlative sialometry and ex vivo histologic-immunohistochemical validation. US revealed reduction in gland volume and an early increase in vascular resistance postradiation. This was accompanied by a reduction in glandular oxygen consumption on PAI. Imaging data correlated strongly with salivary secretion and histologic evidence of acinar damage. The magnitude and kinetics of radiation response were impacted by host immune status, with immunodeficient mice showing early and more pronounced vascular injury and DNA damage response compared to immunocompetent animals. Our findings demonstrate the ability of noninvasive US-PAI to monitor dynamic changes in salivary gland hemodynamics following radiation and highlight the impact of the host immune status on salivary gland radiation injury.

Impact of Age on Disease Progression and Microenvironment in Oral Cancer.

Despite the recognized link between aging and cancer, most preclinical studies in experimental tumor models are conducted with 6- to 8-wk-old rodents. The goal of the present study was to examine the impact of age on tumor incidence, growth, and microenvironmental characteristics in mouse models of head and neck squamous cell carcinoma (HNSCC). Experimental studies were conducted with the 4-nitroquinoline-oxide (4NQO) oral carcinogenesis model and orthotopic FaDu HNSCC xenografts, established in young (7 to 12 wk of age) and old (65 to 70 wk of age) female C57BL/6 mice ( n = 44; 4NQO model) and severe combined immunodeficient mice ( n = 13; HNSCC xenografts). Noninvasive whole body magnetic resonance imaging revealed increased subcutaneous and visceral fat in aging animals of both strains. On histologic examination, a higher incidence ( P < 0.001) of severe dysplasia/invasive squamous cell carcinoma was observed in old mice (92%) as compared with young mice (69%). Old C57BL/6 mice exposed to 4NQO exhibited increased incidence of oral and extraoral (peritoneal masses) neoplasms (42%) versus their young counterparts ( P < 0.05). The incidence of extraoral neoplasms was significantly lower (16%) in the younger cohort. Interestingly, no difference in growth rate and oxygen saturation was observed between orthotopic FaDu xenografts established in old and young severe combined immunodeficient mice. Our observations suggest that host age may have an impact on the growth kinetics and progression of HNSCC in the immunocompetent 4NQO model. Further investigation into the impact of aging on tumor response to preventive and therapeutic intervention is warranted.

Combination of 1alpha,25-dihydroxyvitamin D(3) with dexamethasone enhances cell cycle arrest and apoptosis: role of nuclear receptor cross-talk and Erk/Akt signaling.

Previously we have shown that dexamethasone (DEX) enhances the antitumor activity and ligand binding of the active form of vitamin D, 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), in the murine squamous cell carcinoma model SCC VII/SF. DEX also reduces the hypercalcemia toxicity of 1,25-D(3) treatment. However, the mechanism of the enhanced antitumor activity has not been defined. Here, we demonstrate that both cell cycle arrest and apoptosis were enhanced by DEX, effects that were inhibited by RU486. We also demonstrate that vitamin D receptor (VDR) protein levels were increased by the combination of 1,25-D(3) and DEX above the level observed with 1,25-D(3) treatment alone, whereas protein levels of the heterodimeric partner of VDR, retinoid X receptor, were lower for the combination than for 1,25-D(3) alone. Glucocorticoid receptor protein levels and ligand binding were increased by 1,25-D(3) but not by the combination. Treatment with the combination of 1,25-D(3) and DEX did not result in greater activation of a vitamin D response element-reporter than 1,25-D(3) alone or of a glucocorticoid response element-reporter than DEX alone. Nevertheless, the levels of phospho-Erk1/2 and phospho-Akt, signaling molecules that are modulated in 1,25-D(3)-treated squamous cell carcinoma cells, were reduced by the combination of 1,25-D(3) and DEX more than by either agent alone. These trends were also observed in vivo. Our results suggest the involvement of the Erk and Akt signaling pathways in the antiproliferative effects of the combination of 1,25-D(3) and DEX and that phospho-Erk1/2 and phospho-Akt may be useful markers of response to this combination.

Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis.

We demonstrated that calcitriol has antiproliferative activity in squamous cell carcinoma and prostatic adenocarcinoma and enhances the antitumor activity of platinum-based agents. In this study, we examined whether calcitriol also increases paclitaxel cytotoxicity. The effect of treatment on growth of the murine squamous cell carcinoma (SCCVII/SF) and human prostatic adenocarcinoma (PC-3) was determined by clonogenic assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and monitoring tumor growth. Treatment of SCC or PC-3 cells in vitro with calcitriol prior to paclitaxel significantly reduced clonogenic survival compared with either agent alone. Median-dose effect analysis revealed that calcitriol and paclitaxel interact synergistically. Treatment of SCC or PC-3 tumor-bearing mice with calcitriol prior to paclitaxel resulted in substantially greater growth inhibition than was achieved with either agent alone, supporting the combined use of calcitriol and paclitaxel in the treatment of solid tumors. To explore the molecular basis for the enhanced antitumor activity of this combination, the effect of treatment on p21(Waf-1) (p21), Bcl-2, and poly(ADP-ribose) polymerase expression was evaluated in PC-3. A 72-h pretreatment with calcitriol reduced p21 expression and increased paclitaxel cytotoxicity (measured after 24 h) without evidence of apoptosis [poly(ADP-ribose) polymerase cleavage]. After 48 h, paclitaxel induced apoptosis, the extent of which was increased similarly by pretreatment or concurrent treatment with calcitriol. We therefore propose a model for calcitriol enhancement of paclitaxel cytotoxicity in which the "early" (24 h) effects are schedule dependent and not attributed to enhancement of paclitaxel-induced apoptosis. In contrast, the "delayed" (48-h) enhancement of paclitaxel activity by calcitriol is schedule independent and associated with acceleration of apoptosis.

1,25-Dihydroxycholecalciferol (1,25-D3) inhibits the growth of squamous cell carcinoma and down-modulates p21(Waf1/Cip1) in vitro and in vivo.

1,25-Dihydroxycholecalciferol (1,25-D3) has significant antitumor effects in the murine squamous cell carcinoma (SCC) tumor model in vitro and in vivo. We investigated the basis for this antiproliferative activity and found that, in vitro, 1,25-D3 administration is associated with altered expression of cell cycle regulatory proteins, treatment results in retinoblastoma dephosphorylation, decreased expression of p21(Waf1/Cip1) (p21) mRNA and protein, and increased expression of p27Kip1 (p27) mRNA and protein. Dexamethasone, which acts synergistically with 1,25-D3 to inhibit SCC proliferation, enhanced 1,25-D3-induced down-modulation of p21 without affecting the ability of 1,25-D3 to increase p27 expression. 1,25-D3 did not induce cleavage of poly(ADP-ribose) polymerase. These in vitro data suggest that 1,25-D3 exerts antitumor activity in SCC by perturbing cell cycle progression rather than by inducing apoptosis. In vivo, a 1,25-D3 treatment regimen that results in a decrease in SCC tumor volume is associated with a statistically significant decrease in intratumoral p21 expression. p21 expression is not changed in tumors isolated from control animals or animals treated with a nontherapeutic dose of 1,25-D3. Intratumoral p27 levels were not modulated by 1,25-D3 treatment. Thus, both in vitro and in vivo, 1,25-D3-mediated growth inhibition is associated with p21 down-modulation.

Characterization of a novel Bax-associated protein expressed in hemopoietic tissues and regulated during thymocyte apoptosis.

Members of the Bcl-2 protein family have been implicated as critical intracellular regulators of apoptosis. Most studies of this protein family have utilized transformed and/or transfected cell lines expressing high levels of these proteins. In the current study, we have analyzed normal murine lymphoid cells and tissues and have detected a previously unreported protein of approximately 16 kDa recognized by an anti-Bax Ab. This 16-kDa protein is abundant in hemopoietic tissues of both wild-type and Bax knock-out mice, it can heterodimerize with Bax in normal lymphocytes, and it is dramatically down-modulated in thymocytes in response to apoptotic stimuli. These results suggest that this protein may have antiapoptotic activity and may participate in the regulation of apoptosis in normal lymphocytes.

Down-modulation of a novel Bax-associated protein during apoptosis in normal mature B lymphocytes.

We have recently characterized a novel 16-kDa Bax-associated protein. In this study, we investigate the regulation of this protein's expression during in vitro induction of apoptosis in mature splenic B cells. A panel of biochemically distinct apoptotic stimuli induced the dramatic down-modulation of the 16-kDa protein in B cells; this down-modulation was rapid, and did not require DNA fragmentation. Reciprocally, stimuli that induced protection from apoptosis prevented down-modulation of the 16-kDa protein. These regulatory effects were specific, since Bcl-2 and Bax protein levels were not similarly modulated. Stimuli that reduce expression of the 16-kDa protein may therefore act indirectly to increase the proapoptotic activity of Bax, perhaps by altering Bax binding to other cellular proteins.

In vitro thymocyte maturation is associated with reduced cellular susceptibility to Fas-mediated apoptosis.

We have developed a novel system in which the susceptibility of murine thymocytes to Fas-mediated apoptosis can be modulated. Thymocyte susceptibility to Fas decreases under in vitro culture conditions that promote aspects of thymocyte maturation. The hyporesponsive state is specific for the Fas pathway, since cellular susceptibility to other apoptotic stimuli is not reduced. Hyporesponsiveness is not associated with alterations in the thymocyte subset distribution, decreased expression of full-length Fas protein, or alterations in FADD, Bcl-2, or Bcl-XL expression. Hyporesponsiveness is overcome by increasing the strength of the Fas cross-linking stimulus, leading us to propose that reduced thymocyte susceptibility to apoptosis results from altered Fas signaling. The block in Fas signaling resides proximal to ceramide generation, since Fas-hyporesponsive thymocytes are susceptible to ceramide-induced apoptosis. Further characterization of Fas signaling in these in vitro cultured thymocytes may facilitate the identification of factors regulating the susceptibility of wild-type lymphocytes to Fas.

Fas ligand (CD95L) and B7 expression on dendritic cells provide counter-regulatory signals for T cell survival and proliferation.

Activation of T cells is induced efficiently by dendritic cells (DC), but little is known about the role of DC in the regulation of T cell death. In this study, highly purified DC (DEC-205+, MHC class II(high), B7-1+ [CD80+], B7-2high [CD86high], CD40+, CD11c+) grown from normal mouse bone marrow in granulocyte-macrophage CSF + IL-4 were found to express FasL (CD95L) mRNA by reverse transcriptase PCR and to uniformly express FasL by both flow cytometric and immunocytochemical analyses. These cells, but not DC propagated from FasL-deficient (B6.gld) mice, induced dose-dependent increases in DNA fragmentation in Fas+ Jurkat T cells over 18 h coculture. Addition of mouse Fas-Fc fusion protein at the start of the cultures diminished this effect. Even at high relative concentrations, however, B7-2high DC induced only low levels of DNA fragmentation in Con A or alloactivated splenic T cells, as determined by radio- or spectrofluorometric assays and by in situ nick-end labeling. However, in the presence of CTLA4Ig, a molecule that blocks the B7-CD28 costimulatory pathway, DC that failed to stimulate in primary MLR induced markedly augmented levels of apoptosis in alloactivated T cells. CTLA4Ig treatment also increased the level of DNA fragmentation induced by FasL-deficient DC, indicating the existence of additional potential (Fas-independent) pathways of DC-induced T cell death. These findings suggest that the costimulatory (B7-CD28) and T cell death-inducing pathways may play important counter-regulatory roles in dictating the outcome of (allogeneic) DC-T cell interactions.

Suppression of apoptosis in insect cells stably transfected with baculovirus p35: dominant interference by N-terminal sequences p35(1-76).

Expression of p35 from the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV) suppresses virus-induced apoptosis and promotes virus replication in Spodoptera frugiperda (SF21) cells. To examine the molecular mechanism by which p35 prevents apoptosis in insects, SF21 cells were stably transfected with p35. Neomycin-resistant cell lines that synthesized protein P35 were identified. Stable transfection with p35 protected SF21 cells from apoptosis induced by actinomycin D concentrations that caused apoptotic death of untransfected cells. Cellular expression of p35 also blocked apoptosis induced by infection with p35 null mutants and restored mutant replication to levels comparable to those of wild-type virus. In contrast, stable expression of the mammalian death suppressor bcl-2 failed to block actinomycin D- or AcMNPV-induced apoptosis. Thus, p35 was sufficient to prevent apoptosis, whereas bcl-2 was not, suggesting that the activities of the two nonhomologous death regulators are functionally distinct. Stable expression of the truncation mutant p35(1-76), containing the N terminus of p35, failed to block apoptosis. However, p35(1-76) interfered with p35 antiapoptotic activity, since stably transfected cells underwent apoptosis upon infection with wild-type AcMNPV. Despite normal levels of viral p35 transcription, P35 levels were selectively reduced during infection. Thus, p35(1-76) acted as a dominant inhibitor by directly or indirectly affecting the synthesis or stability of viral P35. These results suggested that the N terminus of P35 constitutes a functional domain which is required to interact with other proteins, possibly host invertebrate death regulators or P35 itself.

The apoptotic suppressor P35 is required early during baculovirus replication and is targeted to the cytosol of infected cells.

The p35 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is required to block virus-induced apoptosis. The trans-dominant activity of p35 suppresses premature cell death and facilitates AcMNPV replication in a cell line- and host-specific manner. To characterize the p35 gene product (P35), a specific polyclonal antiserum was raised. As revealed by immunoblot analyses of wild-type AcMNPV-infected cells, P35 appeared early (8 to 12 h) and accumulated through the late stages of infection (24 to 36 h). Biochemical fractionation of cells both early and late in infection and indirect immunochemical staining demonstrated that P35 localized predominantly to the cytosol (150,000 x g supernatant); comparatively minor quantities of P35 were associated with intracellular membranes. The cytoplasmic localization of P35 was independent of virus infection. The functional significance of the early and late synthesis of P35 was examined by constructing recombinant viruses in which the timing and level of p35 expression were altered. Delaying P35 synthesis by placing p35 under exclusive control of a strong, very late promoter failed to suppress intracellular DNA fragmentation and apoptotic blebbing in most cells. Thus, earlier expression of p35 was required to block virus-induced apoptosis. Site-specific mutagenesis of the p35 promoter demonstrated that low levels of P35 were sufficient to block apoptosis, whereas higher levels were required to maintain wild-type virus gene expression. Consistent with an early role in infection, P35 was also detected in the budded form of AcMNPV. Because of the lack of sequence similarity and its cytosolic targeting, P35 may function in a manner that is mechanistically distinct from other apoptotic regulators, including Bcl-2 and the adenovirus E1B 19-kDa protein.

Interference by PR-bound RNA polymerase with PRM function in vitro. Modulation by the bacteriophage lambda cI protein.

Activation of the weak PRM promoter by cI protein is an essential process in the establishment of lysogeny. Much evidence has accumulated that cI protein binds cooperatively to the operators OR1 and OR2 and that protein at the OR2 site contacts RNA polymerase to facilitate open complex formation at the PRM promoter. We had shown previously in vitro that RNA polymerase situated at the nearby PR promoter could interfere with open complex formation at PRM and that an additional mechanism of PRM activation in vitro involved cI-mediated RNA polymerase exclusion from PR. Here we further characterize this second indirect mode of activation. We demonstrate the addition of cI and inactivation of the PR promoter activate open complex formation at PRM similarly over the temperature range from 37 to 20 degrees C in which the extent of activation decreases from 8- to 2-fold. We also show that the binding of cI protein to OR1 is sufficient to effect an increase in the rate of synthesis of abortive RNA products at PRM. This result is difficult to explain based on direct cI-RNA polymerase contacts alone but is readily interpreted in terms of our previously proposed model involving the exclusion of an interfering RNA polymerase from binding at PR.

Site-specific mutagenesis of the 35-kilodalton protein gene encoded by Autographa californica nuclear polyhedrosis virus: cell line-specific effects on virus replication.

The gene encoding the 35-kDa protein (35k gene) located within the EcoRI-S genome fragment of Autographa californica nuclear polyhedrosis virus (AcMNPV) is transcribed early in infection. To examine its function(s) with respect to virus multiplication, we introduced specific mutations of this early gene into the AcMNPV genome. In Spodoptera frugiperda (SF21) culture, deletion of the 35K gene reduced yields of extracellular, budded virus from 200- to 15,000-fold, depending on input multiplicity. Mutant replication was characterized by dramatically diminished levels of late and very late (occlusion-specific) virus gene expression and premature cell lysis. In contrast, 35K gene inactivation had no effect on virus growth in cultured Trichoplusia ni (TN368) cells. Insertion of the 35K gene and its promoter at an alternate site (polyhedrin locus) restored virus replication to wild-type levels in SF21 culture. Subsequent insertion of 4 bp after codon 81 generated a frameshift mutant that exhibited a virus phenotype indistinguishable from that of 35K deletion mutants and demonstrated that the 35K gene product (p35) was required for wild-type replication in SF21 cells. Mutagenesis also indicated that the C terminus of p35, including the last 12 residues, was required for function. In complementation assays, wild-type virus bearing a functional 35K gene allele stimulated all aspects of 35K null mutant replication and suppressed early cell lysis. These findings indicated that p35 is a trans-dominant factor that facilitates AcMNPV growth in a cell line-specific manner.

RNA polymerase bound to the PR promoter of bacteriophage lambda inhibits open complex formation at the divergently transcribed PRM promoter. Implications for an indirect mechanism of transcriptional activation by lambda repressor.

We demonstrate that RNA polymerase bound at the PR promoter of bacteriophage lambda can repress transcription initiation from the divergently transcribed PRM promoter in vitro. Using abortive initiation and run-off transcription experiments we show that inactivating mutations introduced into either the -10 or -35 regions of PR result in a significant increase in the rate of formation of transcriptionally competent complexes at the PRM promoter. This is due primarily to an increase in the rate constant for the isomerization of closed to open complexes. Gel shift and DNase I footprinting experiments were employed to further define the mechanism by which PR sequences mediate PRM repression. From these assays we were able to conclude that the formation of an open complex at the PR promoter did not exclude RNA polymerase from binding at PRM. Rather, initiation at PRM was impaired because closed complexes must isomerize in the presence of an open complex already situated at the PR promoter. Extensive evidence has been obtained previously indicating that lambda repressor activates transcription directly by contacting RNA polymerase situated at the PRM promoter. Results presented here raise the possibility that an additional mechanism could be operative, whereby lambda repressor indirectly activates PRM transcription by excluding RNA polymerase from the PR promoter.