IL-17-driven intestinal fibrosis is inhibited by Itch-mediated ubiquitination of HIC-5.

Intestinal fibrosis is a major complication in inflammatory bowel diseases, but the regulatory mechanism that inhibits fibrosis remains unclear. Here we demonstrate that Itch-/-myofibroblasts express increased amounts of profibrotic collagen type I and α-SMA in response to IL-17. Mechanistically, we demonstrate that Itch directly binds to HIC-5 and targets it for K63-linked ubiquitination to inhibit IL-17-driven intestinal fibrosis. Reconstitution of Itch-/- myofibroblasts with wild-type Itch but not the Itch-C830A mutant normalized the expression of profibrotic genes. Similarly, shRNA-mediated inhibition of HIC-5 normalized the expression of profibrotic gene expression. Thus, we have uncovered a novel mechanism by which Itch negatively regulates intestinal fibrosis.

Environmental estrogen stimulation of growth and estrogen receptor function in preneoplastic and cancerous human breast cell lines.

BACKGROUND: DDT and polychlorinated biphenyls (PCBs), which are widespread in the ecosystem, can mimic estrogen-mediated cell activities. Thus, they can potentially interfere with many physiologic processes. We compared the effects of organochlorines belonging to the DDT and PCB families, alone and in combination, for their ability to influence the estrogen receptor-mediated activities in preneoplastic breast epithelial cells and breast cancer cells. METHODS: Multiple assay systems requiring functional estrogen receptor were employed to test estrogen-like activity of organochlorine ligands. Two-sided statistical tests were used to compare the data. RESULTS: p,p'-DDT, the predominant form of DDT in the environment, is a more potent estrogen than o,p'-DDT (P<.001), although it is less effective than o,p'-DDT in inhibiting the binding of estradiol (natural estrogen) to estrogen receptor. Among the PCBs, Heptachlor is estrogenic (in transient reporter assays; P< or =.001), whereas Aroclor 1221 and Aroclor 1254, both individually and in combination, are only weakly estrogenic. CONCLUSION: p,p'-DDT is the most effective organochlorine in regulating estrogen receptor-mediated cellular responses. In estrogen receptor-positive breast cancer cells, p,p'-DDT evokes responses by itself and enhances the responses in collaboration with estradiol or o,p'-DDT.

Randomized clinical trial to assess the effectiveness of breast irradiation following lumpectomy and axillary dissection for node-negative breast cancer.

BACKGROUND: Although the conservation management of breast cancer has become a routine method of treatment in most centers, there is still considerable controversy surrounding the ultimate minimum treatment required for node-negative breast cancer to achieve adequate local control. PURPOSE: Our purpose was to assess the value of breast irradiation in reducing breast relapse following conservation surgery for node-negative breast cancer. We attempted to define low-risk groups of women for breast and distant site relapse (i.e., recurrence outside the breast) who might be spared breast irradiation or adjuvant systemic therapy. METHODS: Eight hundred thirty-seven patients were randomly assigned to receive radiation therapy or no radiation therapy following lumpectomy and axillary dissection for node-negative breast cancer. RESULTS: Breast irradiation reduced relapse in the breast from 25.7% in the controls to 5.5% in the irradiated patients. There was no difference in survival between the two groups (median follow-up, 43 months). A low-risk group (less than 5% chance of relapse in the breast without irradiation) could not be defined. Tumor size (greater than 2 cm), age (less than 40 years), and poor nuclear grade were important predictors for breast relapse. Age (less than 50 years) and poor nuclear grade were important predictors for mortality. The presence of ductal carcinoma in situ did not predict breast relapse. CONCLUSIONS: Breast irradiation significantly reduces breast relapse, but it does not influence survival. Important predictors of breast relapse are age, tumor size, and nuclear grade, but not the presence of ductal carcinoma in situ. Age and, in particular, nuclear grade predict survival. IMPLICATIONS: Further follow-up may define an acceptable low-risk group for breast relapse. Until then, we recommend that all patients receive breast irradiation. Systemic adjuvant therapy should be considered for patients with poor nuclear grade tumors.

Synergistic antitumor activity of cisplatin and interleukin 1 in sensitive and resistant solid tumors.

The antitumor activity of cis-diamminedichloroplatinum(II) (cP) and human recombinant interleukin-1 alpha (IL-1 alpha) was studied in RIF-1 and SC VII solid tumor models and in a cP-resistant subline of RIF-1 designated RIF-R1cP. In RIF-1 tumors, clonogenic cell survival after cP plus IL-1 alpha combinations was highly schedule and IL-1 alpha dose dependent. More than additive clonogenic cell kill was seen when cP was given 6 h before, but not 8 h before or at 2-6 h after IL-1 alpha. Time course studies indicated that maximal clonogenic cell killing was achieved within 4-6 h after the cP plus IL-1 alpha combination, with little or no recovery for up to 24 h. In vivo dose-response studies indicated that cP plus IL-1 alpha combinations induced more clonogenic cell kill than cP alone in all three tumor models, and analysis by the median effect principle indicated highly synergistic antitumor activity. Dexamethasone but not indomethacin inhibited the synergistic interaction. IL-1 alpha had no effect on the cytotoxicity of cP in SCC VII cells in vitro, and neither in vitro hypoxia nor in vivo ischemia, induced by clamping tumor blood supply, significantly affected cP clonogenic cell killing. Increased clonogenic cell killing was seen, however, after removal of the clamp, implicating reperfusion events, such as oxyradical stress, as a potential mechanism for increased cP cytotoxicity in SCC VII solid tumors. The data from our model systems provide a rationale for additional work to define the mechanisms of the synergistic antitumor activity of the cP plus IL-1 alpha combination and indicate that IL-1 alpha might be a useful adjunct to increase the clinical efficacy of cP-containing strategies for both sensitive and cP-resistant cancers.

Methotrexate/fluorouracil scheduling influences normal tissue toxicity but not antitumor effects in patients with squamous cell head and neck cancer: results from a randomized trial.

To test the hypothesis that sequential scheduling of methotrexate (MTX) and fluorouracil (FU) produces a synergistic antitumor effect, we randomized 113 patients with recurrent or locally advanced squamous cell carcinoma of the head and neck to receive MTX-FU either 18 hours apart or simultaneously, with leucovorin rescue. There were 100 patients with locally advanced newly presenting disease and 13 patients with recurrence. Excessive toxicity was observed in the first 11 patients who received MTX 250 mg/m2 administered intravenously (IV) and leucovorin at 36 hours, therefore all subsequent patients received MTX 200 mg/m2 administered IV and leucovorin at 24 hours. FU 600 mg/m2 IV was administered to all patients, and treatment was given on days 1 and 8 of 21-day cycles. The treatment groups were well balanced for known prognostic variables. The response rate was 47.3% (26 of 55) for simultaneous v 44.8% (26 of 58) for sequential therapy. These results exclude a 20% difference in response rate favoring sequential therapy at P = .04. There was no observed difference in survival between the two treatment arms (P = .55) with a minimum follow-up of 8 months. Toxicity was greater in patients who received sequential therapy, and the difference was confined to the gastrointestinal (GI) tract. A comparison of the distribution in maximum Eastern Cooperative Oncology Group (ECOG) toxicity scores during chemotherapy for the two treatment groups showed greater stomatitis (P = .001), diarrhea (P = .04), and overall toxicity (P = .02) for sequential treatment without an observed difference in bone marrow toxicity. The results of this trial indicate that sequential MTX-FU is not superior to simultaneous therapy for the treatment of patients with head and neck cancer. Biochemical modulation of MTX-FU by drug scheduling may occur in vivo and may be organ specific.

Potentiation of hydroxyurea cytotoxicity in human chronic myeloid leukemia cells by iron-chelating agent.

The effect of hydroxyurea (HU) was studied in 15 cases of human chronic myeloid leukemia cells (CML) in combination with iron-chelating agent, 2,2-bipyridine (bipyridine). The extent of (3H)thymidine incorporation in CML cells in vitro was taken as an index for the measurement of cytotoxicity. In the present study, we observed a potentiation in HU cytotoxicity by hydrophobic iron-chelator bipyridine (p less than 0.001) resulting in complete DNA biosynthesis inhibition. Both HU and bipyridine were used at a relatively non-toxic concentrations of 10(-4) M and 10 micrograms/ml, respectively. This inhibition was found to be partially reversible and a partial protective action by iron is also demonstrated. The various other metal chelators failed to sensitise CML cells to HU cytotoxicity. Implications with respect to the efficacy in cancer treatment is discussed.

Reversal of natural resistance to bouvardin (NSC 259968) in sarcoma 180 cells in vitro and in vivo by verapamil.

Reversal of natural resistance to bouvardin (NSC 259968) has been attained in vitro and in vivo, by the calcium influx blocker verapamil in sarcoma 180 cells insensitive to bouvardin. Verapamil increased the in vitro lethality of the tumor cells following exposure with cells for 1 and 3 h as a result of the cytotoxic effect of bouvardin. Similar results were observed in vivo when the tumor cells were exposed to verapamil and then treated with bouvardin, showing a significant percent increase in the lifespan to 30% and 45%. This suggested that this calcium channel blocker had interacted with tumor cell membrane to modulate the response of the cells, and make them more amenable to the drug action.

Radioresistance in murine solid tumors induced by interleukin-1.

Interleukin-1 (IL-1) has radioprotective activity in hematopoietic lineages and in other normal cell renewal systems, but little is known about the effects of IL-1 alpha on the radiosensitivity of tumor cell populations. The present studies were conducted to investigate the effects of IL-1 alpha on the radiosensitivity of clonogenic cells in RIF-1 and SCC-7 tumors. Radioresistance was detected within 2-4 after administration of IL-1 alpha (0.5 micrograms/mouse, ip) and characterized by increases in D(o), Dq, alpha/beta and SF2. This radioresistance was similar to that seen in tumors rendered totally hypoxic before X irradiation. Tirapazamine, a hypoxic cell cytotoxin, and IL-1 alpha had synergistic schedule-dependent antitumor activity in vivo, suggesting that IL-1-induced radioresistance in vivo is due to hypoxia. Radioresistance induced by IL-1 alpha was transient, and the data suggested reoxygenation within 12 h. In vitro, IL-1 alpha had no direct effect on the radiosensitivity of SCC-7 cells in tissue culture under aerobic conditions. However, an increase in D(o), alpha/beta and SF2 was seen in clonogenic tumor cells from primary cultures treated with IL-1 alpha under aerobic conditions. Superoxide dismutase and catalase prevented the induction of radioresistance by IL-1 alpha in vitro, suggesting that oxidative responses from tumor macrophages after administration of IL-1 alpha may be responsible for induced radioresistance by IL-1 in vitro. Although oxidant stress induced by IL-1 and in vitro in our models, the mechanisms by which such responses modulate tumor radiosensitivity in vivo and in vitro are likely quite different.

The value of endorectal ultrasonography in the follow-up of intracavitary radiation treated early rectal cancer.

In selected patients with early rectal cancer, intracavitary radiation is a successful treatment. Endorectal ultrasound has proved an accurate method for staging and selecting such cancers for treatment. The value of endorectal ultrasound in the follow-up of patients with intracavitary radiation has not been previously assessed. Between 1989 and 1991, 30 patients treated at the Hamilton Regional Cancer Centre with intracavitary radiation were assessed by endorectal ultrasound. The mean age was 65 +/- 12 years with a range of 37-78 years. There were 17 males and 13 females. All patients were treated with curative intent. The dose of radiation administered was 8963 +/- 1506 cGy over 3.5 +/- 0.7 fractions. No patient received supplemental iridium implantation. Thirty-seven endorectal ultrasounds were carried out in 30 of the intracavitary radiation treated patients. Clinical findings (digital, sigmoidoscopic, and histology) were compared with the radiologist's interpretation of the endorectal ultrasounds. Using a 2 x 2 table accepting the clinical findings as the 'Gold Standard', the sensitivity of endorectal ultrasound was 71%, the specificity 61%, the positive predictive value 53%, the negative predictive value 78% with an overall accuracy of 75%. We conclude that endorectal ultrasound in the routine follow-up of patients treated with intracavitary radiotherapy for carcinoma of the rectum is questionable.

Sensitization of P388 murine leukemia cells to hydroxyurea cytotoxicity by hydrophobic iron-chelating agents.

The potentiation in the cytotoxic effect of antineoplastic agent hydroxyurea (HU) from the non-toxic concentration on P388 murine lymphocytic leukemia cells is observed with the hydrophobic iron-chelating agents, 2,2-bipyridine (bipyridyl) and 1,10-phenanthroline in a concentration and time dependent manner. However EDTA, EGTA, 2,3-dihydroxybenzoic acid (2,3-DBA), Rhodotorulic acid, 8-hydroxyquinoline (8-Hq), catechin and Desferal, and other chelators failed to show sensitization of these tumor cells towards HU. The potentiation in the cytotoxic activity of HU in combination with the bipyridyl was found to be reversible but was maintained when the drug exposed cells were washed and retreated with the non-toxic concentrations of HU or bipyridyl. The presence of Fe++ blocked completely the cytotoxic action of HU alone and a combination with iron-chelator, and partially reversed the inhibition induced by HU and bipyridyl. These findings suggest that the hydrophobic iron-chelators affect the membrane-mediated transfer, localisation and transient intracellular chelatable iron pools. As a consequence of this, the regulatory role mediated by iron on the overall activity of ribonucleotide reductase enzyme is disturbed leading to a conclusive imbalance in DNA biosynthesis.

The influence of age on the management of anal cancer.

The standard treatment for anal cancer is combination chemo-radiotherapy. Management decisions such as radical chemotherapy, resective surgery for poor response or relapse are frequently modified by age-associated comorbid factors. Between 1980 and 1990, our regional cancer center serving a population of 1.8 million saw 78 patients with squamous carcinoma of the anus. We have compared patients who were younger than 65 years (n = 38) with those older than 65 years (n = 38). The mean +/- standard deviation age for the whole cohort was 65 +/- 12 years, with a ratio of 2 females to each male presenting. Fewer of the elderly age group had major surgery (26% vs. 42%) (p = 0.03), and fewer suffered no toxicity (42% vs. 26%) (p = 0.03). However, 61% of the under-65-year age group are alive disease-free vs. 26% of the elderly group (p = 0.03). Similarly, only 18% of the under-65-year group died with disease compared with 37% of the elderly group (b = 0.03). For the series as a whole, the crude mortality was 42%, with 27% dying of their disease. The stage distribution, and the amount of radiotherapy or chemotherapy administered was not age-specific, but younger patients had more surgery and suffered more toxicity, with a greater proportion remaining alive and disease-free, and fewer dying of their disease. These data suggest that a more aggressive multi-modality approach in the elderly may improve disease response and survival.

Tween-80 alters natural resistance of sarcoma-180 to bouvardin (NSC 259968).

Sarcoma-180 tumour (S-180) exhibits natural resistance to bouvardin (NSC 259968), a protein synthesis inhibitor that also inhibits RNA and DNA synthesis when administered over a range of non-toxic doses using 0.9% NaCl as a vehicle. However, using Tween-80 as a vehicle, there is a substantial enhancement of cytotoxicity and a subsequent increase in the life span of animals bearing the S-180 tumour. This observation was substantiated in vitro by exposing S-180 cells to 10(-6)M bouvardin in the presence and absence of Tween-80. Bouvardin, 10(-6)M, in the absence of Tween-80, inhibited the incorporation of [3H]uridine by 46%, whereas the presence of Tween-80 resulted in a 66% inhibition of uridine incorporation.

Augmentation of hydroxyurea cytotoxicity by sintamil in human chronic myeloid leukemia cells.

The in vitro effect of sintamil, as a modulator alone and in combination with hydroxyurea (HU), on cytotoxicity was studied in 16 cases of human chronic myeloid leukemia (CML). We investigated the cytotoxicity of the drugs as a function of the exposure dose (HU, 10(-4) M; sintamil, 10 micrograms/ml) and the exposure time (1 h) to the agent. Cytotoxicity was evaluated as the inhibition of incorporation of [3H-methyl]thymidine in the nucleic acids of CML cells. Cytotoxicity of HU was greatly enhanced (P less than 0.001) by 1 h exposure of the CML cells to sintamil. The present data indicate that sintamil potentiates the cytotoxic activity of HU in CML cells.

Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC.

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3'-untranslated region (3'-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

NPM-ALK signals through glycogen synthase kinase 3ß to promote oncogenesis.

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3ß as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3ß. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3ß (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3ß levels. Phosphorylation of pS(9)-GSK3ß by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3ß substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3ß. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3ß inhibition. Furthermore, stable knockdown of GSK3ß conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3ß and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3ß and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3ß by PI3K/AKT. The subsequent inhibition of GSK3ß activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3ß as a mediator of NPM-ALK oncogenesis.

Intracavitary radiation for rectal carcinoma.

Thirty-five patients with low-lying rectal adenocarcinoma have been treated with intracavitary radiation (Papillon's technique). Twenty-three were treated for cure and 12 for palliation. The indications for curative intracavitary radiation were mobile polypoid tumors, less than 3 cm in diameter, with Broder's Grades I and 2 differentiation lying less than 11 cm from the anal verge. Doses between 2 000 and 4 000 cGy were delivered to a total of 7 000 to 20 000 cGy with complete resolution of the tumors. Eighty-seven per cent in the curative group are alive and well up to 42 months after treatment with a minimum follow-up of six months. Of the 23 patients treated for cure, three patients had recurrences within 18 months of therapy. Two of the three patients are alive following surgery. The third patient died in the postoperative period. The results of intracavitary radiation are comparable to ablative surgery and avoid a permanent colostomy. Age, frailty, or other medical conditions do not preclude this treatment. Anesthesia and hospitalization are not required. This method can also be used for palliation of recurrent tumors and in patients who are unsuitable for surgery.

Cell surface alterations in murine leukaemia P388 adriamycin-resistant cells: studies on lectin-induced agglutination and rearrangement of lectin receptors.

Cell surface alteration was studied in a subline of murine lymphocytic leukaemia resistant to the broad-spectrum anticancer agent adriamycin (P388/ADR) employing concanavalin A(Con A)-induced agglutination and rearrangement of lectin receptors. Con A induced more agglutination of P388/ADR when compared to the drug-sensitive parental cell line (P388/S). Studies on the redistribution of Con A and Ricinus communis agglutinin-I revealed a high percentage of P388/ADR showing internalized fluorescence, while a majority of P388/S displayed a uniform distribution of fluorescence on the cell surface.