Disruption of transforming growth factor-beta signaling through beta-spectrin ELF leads to hepatocellular cancer through cyclin D1 activation.

Transforming growth factor-beta (TGF-beta) signaling members, TGF-beta receptor type II (TBRII), Smad2, Smad4 and Smad adaptor, embryonic liver fodrin (ELF), are prominent tumor suppressors in gastrointestinal cancers. Here, we show that 40% of elf(+/-) mice spontaneously develop hepatocellular cancer (HCC) with markedly increased cyclin D1, cyclin-dependent kinase 4 (Cdk4), c-Myc and MDM2 expression. Reduced ELF but not TBRII, or Smad4 was observed in 8 of 9 human HCCs (P<0.017). ELF and TBRII are also markedly decreased in human HCC cell lines SNU-398 and SNU-475. Restoration of ELF and TBRII in SNU-398 cells markedly decreases cyclin D1 as well as hyperphosphorylated-retinoblastoma (hyperphosphorylated-pRb). Thus, we show that TGF-beta signaling and Smad adaptor ELF suppress human hepatocarcinogenesis, potentially through cyclin D1 deregulation. Loss of ELF could serve as a primary event in progression toward a fully transformed phenotype and could hold promise for new therapeutic approaches in human HCCs.

RING finger-dependent ubiquitination by PRAJA is dependent on TGF-beta and potentially defines the functional status of the tumor suppressor ELF.

In gastrointestinal cells, biological signals for transforming growth factor-beta (TGF-beta) are transduced through transmembrane serine/threonine kinase receptors that signal to Smad proteins. Smad4, a tumor suppressor, is often mutated in human gastrointestinal cancers. The mechanism of Smad4 inactivation, however, remains uncertain and could be through E3-mediated ubiquitination of Smad4/adaptor protein complexes. Disruption of ELF (embryonic liver fodrin), a Smad4 adaptor protein, modulates TGF-beta signaling. We have found that PRAJA, a RING-H2 protein, interacts with ELF in a TGF-beta-dependent manner, with a fivefold increase of PRAJA expression and a subsequent decrease in ELF and Smad4 expression, in gastrointestinal cancer cell lines (P < 0.05). Strikingly, PRAJA manifests substantial E3-dependent ubiquitination of ELF and Smad3, but not Smad4. Delta-PRAJA, which has a deleted RING finger domain at the C terminus, abolishes ubiquitination of ELF. A stable cell line that overexpresses PRAJA exhibits low levels of ELF in comparison to a Delta-PRAJA stable cell line, where ELF expression is high compared to normal controls. The alteration of ELF and/or Smad4 expression and/or function in the TGF-beta signaling pathway may be induced by enhancement of ELF degradation, which is mediated by a high-level expression of PRAJA in gastrointestinal cancers. In hepatocytes, half-life (t(1/2)) and rate constant for degradation (k(D)) of ELF is 1.91 h and 21.72 min(-1) when coupled with ectopic expression of PRAJA in cells stimulated by TGF-beta, compared to PRAJA-transfected unstimulated cells (t(1/2) = 4.33 h and k(D) = 9.6 min(-1)). These studies reveal a mechanism for tumorigenesis whereby defects in adaptor proteins for Smads, such as ELF, can undergo degradation by PRAJA, through the ubiquitin-mediated pathway.

Age-related spontaneous lacrimal keratoconjunctivitis is accompanied by dysfunctional T regulatory cells.

In both humans and animal models, the development of Sjögren syndrome (SS) and non-SS keratoconjunctivitis sicca (KCS) increases with age. Here, we investigated the ocular surface and lacrimal gland (LG) phenotype of NOD.B10.H2b mice at 7-14, 45-50, and 96-100 weeks. Aged mice develop increased corneal permeability, CD4+ T-cell infiltration, and conjunctival goblet cell loss. Aged mice have LG atrophy with increased lymphocyte infiltration and inflammatory cytokine levels. An increase in the frequency of CD4+Foxp3+ T regulatory cells (Tregs) was observed with age in the cervical lymph node (CLN), spleen, and LG. These CD4+CD25+ cells lose suppressive ability, while maintaining expression of Foxp3 (forkhead box P3) and producing interleukin-17 (IL-17) and interferon-γ (IFN-γ). An increase of Foxp3+IL-17+ or Foxp3+IFN-γ+ cells was observed in the LG and LG-draining CLN. In adoptive transfer experiments, recipients of either purified Tregs or purified T effector cells from aged donors developed lacrimal keratoconjunctivitis, whereas recipients of young Tregs or young T effector cells failed to develop disease. Overall, these results suggest inflammatory cytokine-producing CD4+Foxp3+ cells participate in the pathogenesis of age-related ocular surface disease.

Extremely high production of thymidine by TNF-susceptible L929 cells.

We confirmed with the use of crystal violet bioassay the high susceptibility of mouse L929 cells to the cytotoxic action of recombinant tumor necrosis factor-alpha (TNF). However, when a [3H]thymidine release assay was used for the same purpose, we found that [3H]thymidine uptake by the L929 cells, in contrast to low-malignant TNF-resistant spontaneously transformed Syrian hamster embryo cells of the STHE strain, was significantly reduced (P < 0.001). To investigate the mechanism of the low incorporation of [3H]thymidine in L929 cells the culture media from intact L929 cells was used for competition experiments with [3H]thymidine incorporation in the STHE target cells. The undiluted supernatant from L929 cells significantly (up to 83-97%) reduced [3H]thymidine uptake by the STHE cells. Fifty percent inhibition of [3H]thymidine uptake was achieved at L929 supernatant dilutions up to 1:8 (in 4 h incubation), up to 1:16 (in 20-42 h incubation) and even up to 1:32 (in 42 h incubation). The same high level of inhibition of [3H]thymidine uptake by STHE cells was seen with a commercial specimen of a thymidine (Sigma) at a concentration near 500 ng/ml. Thus, we conclude that a resistance of L929 cells to [3H]thymidine uptake is related to their unusually high production of cold thymidine.

In vivo blocking of SV40 virus-induced antitumor resistance by Syrian hamster sera from early stages of primary SV40 carcinogenesis.

The blocking of specific SV40-induced resistance of hamsters by the sera collected from 12 individual hamsters infected with SV40 when newborn was studied at different periods of primary virus-induced carcinogenesis. The serum samples were collected at the following periods of primary carcinogenesis: during the latent period (60 days after virus inoculation), at the day of primary tumor appearance, and 19-36 and 45-57 days after primary tumor appearance. For detection of the blocking activity of the collected sera the cells of transplantable SV40 test-tumor were pretreated in vitro with these sera and with control normal hamster sera, and then used for challenge in vivo in SV40-immunized and normal hamsters. With the use of such method, as a rule, no blocking activity of the serum samples collected at any time after the tumor appearance was observed. However, the sera obtained from 7 out of 12 of these hamsters during the latent period significantly decreased the resistance index of animals challenged in transplantation test with the serum pretreated tumor cells. Possible explanations of these findings are discussed.

CD8⁺ cells regulate the T helper-17 response in an experimental murine model of Sjögren syndrome.

This study investigated the regulatory function of CD8⁺ cells in T helper-17 (Th17) cell-mediated corneal epithelial barrier disruption that develops in a murine desiccating stress (DS) model that resembles Sjögren syndrome. CD8⁺ cell depletion promoted generation of interleukin-17A (IL-17A)-producing CD4⁺ T cells via activation of dendritic cells in both the ocular surface and draining cervical lymph nodes in C57BL/6 mice subjected to DS. T-cell-deficient nude recipient mice receiving adoptively transferred CD4⁺ T cells from CD8⁺ cell-depleted donors exposed to DS displayed increased CD4⁺ T-cell infiltration and elevated IL-17A and CC-chemokine attractant ligand 20 levels in the ocular surface, which was associated with greater corneal barrier disruption. Enhanced DS-specific corneal barrier disruption in CD8-depleted donor mice correlated with a Th17-mediated expression of matrix metalloproteinases (MMP-3 and MMP-9) in the recipient corneal epithelium. Co-transfer of CD8⁺CD103⁺ regulatory T cells did not affect the ability of DS-specific pathogenic CD4⁺ T cells to infiltrate and cause ocular surface disease in the nude recipients, showing that CD8⁺ cells regulate the efferent arm of DS-induced immune response. In summary, CD8⁺ regulatory cells suppress generation of a pathogenic Th17 response that has a pivotal role in DS-induced disruption of corneal barrier function.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13.

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK- populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Resistance of in vivo-selected spontaneously transformed cells and Rous sarcoma virus-transformed cells to macrophage-mediated cytotoxicity.

The cytotoxic activity (CTA) of activated peritoneal macrophages (MP) on variant lines of Syrian hamster embryo (HE) cells of differing malignant characteristics was studied. The target cells were a line of low-malignant cells resulting from spontaneous transformation of HE cells in vitro (STHE strain), and malignant variants selected from them in vivo (STHE-LM-4, STHE-LM-8, and STHE-75/18 strains). In addition, we used cells of the HET-SR-1 strain; these are HE cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain, RSV-SR), or the TU-SR strain induced by RSV-SR in vivo. Thioglycollate-elicited peritoneal MP from Syrian hamsters were activated in vitro with bacterial levan, LPS or MDP and used as effector cells. MP-mediated cytolysis was determined by means of a 42-h radioactivity release assay with 3H-thymidine-labeled target cells. We found that only the parental STHE cells were susceptible towards fully-activated MP-mediated CTA. All three of the in vivo-selected malignant variants of the STHE cell sublines, as well as the tumorigenic RSV-SR transformants, were resistant to cytolysis by activated MP. Non-activated thioglycollate-elicited MP did not lyse any of the tumor cells studied.

Serum activity inhibiting specific simian virus 40-induced transplantation resistance and its correlation with primary SV40 tumors appearance in hamsters.

Using the modified technique of transplantation test, ITR serum activity was found in most (14 out of 21) individual hamster sera obtained during the latent period of primary SV40 carcinogenesis (60 days after virus infection when newborn). On the other hand, as a rule, no ITR activity was observed in the sera of the same hamsters after tumor appearance and during their growth. ITR activity rapidly disappeared from sera of hamsters neonatally infected with SV40 after their successful immunization with the same virus during the latent period. There appears to be a correlation between the presence of ITR serum factor during the latent period and the subsequent primary SV40 tumor appearance in hamsters.

Resistance of spontaneously transformed Syrian hamster embryo cells and their malignant variants to cytotoxic activity of recombinant tumor necrosis factor-alpha.

To study the possible role of the host macrophages in the selection of tumor cells and tumor progression, a series of Syrian hamster tumor cell lines all originating from a single spontaneously transformed Syrian hamster embryo cell line (STHE strain) have been established. These STHE tumor cell variants, selected either in vitro with resident and lipopolysaccharide-activated macrophages or in vivo, differ in tumorigenic and metastatic activity. The selected malignant STHE cells become resistant to cytotoxic activity of activated peritoneal macrophages and of exogenous hydrogen peroxide (H2O2). Since activated macrophages are a known source for both cytotoxic agents H2O2 and tumor necrosis factor (TNF), the purpose of the present study was to define the sensitivity of the STHE tumor cell lines to a direct cytotoxic activity mediated by recombinant TNF-alpha in an attempt to understand the role of the cytokine in in vitro selection of a malignant STHE cells by activated macrophages. The spontaneously transformed STHE cells (selected in vivo and in vitro) as well as the hamster embryo cells transformed in vitro by a tumorigenic Rous sarcoma virus (Schmidt-Ruppin strain) were used as targets. TNF-alpha-sensitive mouse L929 cells were included in the study as a positive control. Sensitivity of actinomycin D-pretreated target cells studied for cytotoxic activity of a recombinant TNF-alpha was examined over 21 h with a crystal violet dye assay. It was found that, in contrast to L929 cells, the spontaneously transformed STHE cells as well as tumorigenic Rous sarcoma virus hamster embryo transformants, were all significantly resistant to the TNF-alpha-mediated cytolysis. This indicates that TNF-alpha is not the single factor responsible in in vitro selection of malignant STHE cell variants by activated macrophages. It appears that H2O2 is involved in the selection of the hamster macrophage-resistant STHE tumor cells.