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The association of human breast cancer with sequences similar to the mouse mammary tumor virus (MMTV) has been shown, but convincing evidence for the presence of viral particles in breast tumors has been lacking. We have described the complete proviral structure of a retrovirus in human breast cancer. This provirus, designated as human mammary tumor virus (HMTV), was 95% homologous to MMTV and revealed features of a replication-competent virus. We have therefore investigated the production of viral particles in primary cultures of human breast cancer (MSSM). Cells isolated from ascites or pleural effusions of patients with metastatic breast cancer contained viral sequences in their DNA, expressed Env protein, and showed retroviral particles by electron microscopy. Viral particles from culture media exhibited morphologic features of β-retroviruses sedimenting at buoyant densities of 1.12 to 1.18 g/mL in sucrose gradients and showed reverse transcriptase activity. cDNA sequences from virion RNA were synthesized, amplified, and sequenced and all the virion genes were detected and 70% of the virion RNA was sequenced. The sequence homologies were, respectively, 85% to 95% compared with the MMTV and HMTV proviruses we have previously described. These results clearly show that breast cancer cells in primary cultures produced HMTV viral particles that are similar to the mouse virus and which may play a role in human breast cancer pathogenesis. [Cancer Res 2007;67(18):8960–5]

Supplementary Table 1 from Characterization of Viral Particles Isolated from Primary Cultures of Human Breast Cancer Cells

The accuracy of timing across a seismic network is important for locating earthquakes as well as studies that use phase-arrival information (e.g., tomography). The Global Seismographic Network (GSN) was designed with the goal of having reported timing be better than 10 ms. In this work, we provide a brief overview of how timing is kept across the GSN and discuss how clock-quality metrics are embedded in Standard for Exchange of Earthquake Data records. Specifically, blockette 1001 contains the timing-quality field, which can be used to identify time periods when poor clock quality could compromise timing accuracy. To verify the timing across the GSN, we compare cross-correlation lags between collocated sensors from 1 January 2000 to 1 January 2020. We find that the mean error is less than 10 ms, with much of the difference likely coming from the method or uncertainty in the phase response of the instruments. This indicates that timing across the GSN is potentially better than 10 ms. We conclude that unless clock quality is compromised (as indicated in blockette 1001), GSN data’s timing accuracy should be suitable for most current seismological applications that require 10 ms accuracy. To assist users, the GSN network operators have implemented a “gsn_timing” metric available via the Incorporated Research Institutions for Seismology Data Management Center that helps users identify data with substandard timing accuracy (the 10 ms design goal of the GSN).

This Phase 1/2, dose-escalating study of rigosertib enrolled 22 patients with higher-risk myelodysplastic syndromes (MDS) (n=9) and acute myeloid leukemia (AML; n=13) who had relapsed or were refractory to standard therapy and for whom no second-line therapies were approved. Patients received 3- to 7-day continuous intravenous infusions of rigosertib, an inhibitor of Ras-effector pathways that interacts with the Ras-binding domains, common to several signaling proteins including Raf and PI3 kinase. Rigosertib was administered at doses of 650-1700mg/m2/day in 14-day cycles. Initial dose escalation followed a Fibonacci scheme, followed by recommended phase 2 dose confirmation in an expanded cohort. Rigosertib was well tolerated for up to 23 cycles, with no treatment-related deaths and 18% of patients with related serious adverse events (AEs). Common AEs were fatigue, diarrhea, pyrexia, dyspnea, insomnia, and anemia. Rigosertib exhibited biologic activity, with reduction or stabilization of bone marrow blasts and improved peripheral blood counts in a subset of patients. Ten of 19 evaluable patients (53%) demonstrated bone marrow/peripheral blood responses (n=4 MDS, n=1 AML) or stable disease (n=3 MDS, n=2 AML). Median survival was 15.7 and 2.0 months for responders and non-responders, respectively. Additional studies of rigosertib are ongoing in higher-risk MDS (NCT00854646).

Cancer is usually diagnosed late in rural Africa leading to incurability and abbreviated survival. Many curable cancers present on the body surface, often recognizable early by laymen as suspicious, justifying professional referral. Cancer diagnoses in two randomly chosen Tanzanian villages were compared after conventional dispensary self-referral vs. proactive visits in the home. Village navigators organized trips for professional consultation. In the control village 21% were self-referred, 20% of them were sent on as suspicious, 78% had cancer (8% in men) 0.9% of the village population. In the intervention village 99% were screened, 14% were referred for professional opinion, 93% had cancer (32% in men) 1.6% (p < 0.01 compared with control village). In the second and third years similar activity yielded 0.5% cancer annually in the control village for a 3 year total of 1.86% whereas interventional villagers had 1.4% and 0.6% cancer for a 3 year total of 3.56% (p < 0.001). Downstaging was recognized in the second and third years of intervention from 23 to 51 to 74% Stages I and II (p < 0.001) but in the control village Stages I and II changed from 11% to 22% to 37% (p = NS). The greatest downstaging occurred in breast and cervix cancers.

Rigosertib (ON 01910.Na) is an inhibitor of the phosphoinositide 3-kinase and polo-like kinase pathways that induces mitotic arrest and apoptosis in neoplastic cells, while sparing normal cells. Our purpose is to summarize the clinical activity and safety of intravenous (IV) rigosertib delivered by an external ambulatory infusion pump in patients with refractory anemia with excess blasts-1, -2, or, -t myelodysplastic syndromes (MDS) following prior treatment with DNA methyltransferase (DNMT) inhibitors. A total of 39 patients with MDS who fulfilled these criteria were enrolled in four phase 1-2 clinical trials of IV rigosertib. Thirty five (88%) had higher risk disease according to the Revised International Prognostic Scoring System. Median overall survival for this group of 39 patients was 35 weeks. Of 30 evaluable patients with follow-up bone marrow biopsies, 12 (40%) achieved complete (n = 5) or partial (n = 7) bone marrow blast responses. In addition, 15 patients achieved stabilization of bone marrow blasts. One patient with a complete bone marrow response also achieved a complete cytogenetic response. A second patient with stable bone marrow blasts achieved a partial cytogenetic response. Two of the responding patients and three patients with stable disease had hematological improvements. Rigosertib-induced bone marrow blast decreases and stability appeared to be predictive of prolonged survival. IV rigosertib had a favorable safety profile without significant myelosuppression. Most common drug-related toxicities included fatigue, diarrhea, nausea, dysuria, and hematuria. In summary, IV rigosertib is well tolerated and has clinical activity in patients with higher risk MDS following DNMT inhibitor treatment. A multinational pivotal phase 3 randomized clinical trial of rigosertib versus best supportive care for patients with MDS with excess blasts following prior treatment with DNMT inhibitors (ONTIME: ON 01910.Na Trial In Myelodysplastic SyndromE) has recently completed enrollment.

Forty percent of American women's breast cancers contain HMTV, a betaretrovirus 90-98% homologous to MMTV, the causative agent for breast cancer in mice. HMTV is reverse-transcribed and integrated into chromosomal DNA of human breast cells. MMTV contamination of our analyses has been definitively excluded. In 8% of unselected American mothers HMTV is found in cells in milk, but in 21% of milks from women previously biopsied for unconfirmed cancer suspicion. In breasts biopsied for suspicion of cancer before a later diagnosis of breast cancer, we see suggestive morphologic changes if the subsequent breast cancer is HMTV+. HMTV is infectious in vitro for B and T lymphocytes, dendritic cells, and human mammary epithelial cells. Infecting MCF-10 breast cell lines with HMTV produces molecular changes of epithelial-mesenchymal transition with upregulation of vimentin ,and downregualtion of E-cadherin. The excess of breast cancer in western European countries and their former colonies (age standardized rates of 47 to 92 per 100,000 per year) compared to Asian incidence (29 to 43 ASR/y) can be explained by excess HMTV-related breast cancer incidence. In 7 West European, American and Oceania countries, 30 to 60% contain HMTV, while in 5 Asian nations HMTV in breast cancers ranges from 0 to 22% Different indigenous murine species with disparate MMTV burdens parallel these findings: mus domesticus in the West with much MMTV in its genome, and m. castaneus or m.musculus in the East with less. Ancient contamination of the food chain, and current lactational transfer perpetuate infection. HMTV poses a new and challenging dimension to breast cancer research involving diagnosis, molecular mechanisms, epidemiology, therapy and prevention. Citation Format: Holland JF, Jaffer S, Melana S, Nartey T, Gnjatic S, Pogo BG-T. Human mammary tumor virus, HMTV, is an MMTV-adapted human pathogen. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-06-01.

Objective Human mammary tumor virus (HMTV) is 90% to 98% homologous to mouse mammary tumor virus, the etiological agent of mammary tumors in mice. Human mammary tumor virus sequences were found in 40% of the breast cancers studied in both American and Australian women. In addition, 10% of endometrial carcinomas studied in Australian women also contained HMTV sequences. We have explored the possibility that endometrial cancer of American women may also contain HMTV. Methods/Materials Nested polymerase chain reactions, radioactive internal probing, and sequencing were used to establish the presence of unique nucleotide sequences of HMTV in human genomic DNA. The genomic DNAs were tested to guarantee that they were free of murine DNA. Immunohistochemistry with a monoclonal antibody specific for HMTV envelope protein demonstrated that HMTV sequences were translated. Results Thirteen (23.2%) of 56 of the endometrial cancers studied contained HMTV sequences and proteins. Human mammary tumor virus sequences and protein were not detected in the 33 normal endometria studied. Conclusion Human mammary tumor virus, an agent with high homology to mouse mammary tumor virus, was found in 23.2% of the endometrial cancers studied, thus opening the possibility of a pathogenic role.

Background There is substantial evidence that a virus homologous to mouse mammary tumor virus (MMTV) may have a role in human breast cancer. The present study indicates that those who developed breast cancer associated with an MMTV-like virus had this virus in their non-cancerous breast tissues years before the cancer developed. Methods Polymerase chain reaction (PCR) techniques and sequencing were used to identify MMTV-like envelope gene sequences (MMTV-like env sequences) in Australian benign breast biopsy specimens from women who several years later developed breast cancer. Murine contamination was excluded by stringent laboratory procedures, and the absence of intracisternal A particle sequences and mitochondrial cyclooxygenase sequences. Results MMTV-like env sequences (also called HMTV sequences to denote their source) were found in 9 of 25 breast cancer specimens (36%). Among 25 non-cancerous breast biopsies of these same patients taken 1 to 11 years earlier, six contained MMTV-like sequences (24%). Five of the six were among the nine virally-associated breast cancers. In two pairs of specimens, benign and malignant, env sequences were 97% identical. Conclusions The identification of MMTV (MMTV-like) sequences in breast tissues prior to the development of MMTV positive breast cancer fulfills a key criterion for a possible causal role for the MMTV-like virus in human breast cancer.

Mouse mammary tumor virus (MMTV) has been proven to induce mammary cancer in mice. MMTV-like env gene sequences have been detected in one-third of the human breast tumors studied. The whole proviral structure with 95% homology to MMTV was found in two human breast tumors and was designated as human mammary tumor virus (HMTV). HMTV viral particles with betaretroviral features have been isolated. In addition, a retrovirus called human betaretrovirus (HBRV), homologous to the mentioned retroviruses, has been isolated from tissues of patients with primary biliary cirrhosis. In this report, the expression of HMTV envelope (Env) and capsid (Ca) was detected in 10 primary cultures of human breast cancer containing HMTV sequences (MSSM) by Western blot and fluorescence activated cell sorting (FACS), using a panel of antibodies against HMTV Env, HBRV Env and Ca and the MMTV Env Gp36 and Ca P27 proteins. By contrast, HMTV proteins did not react with antibody against the MMTV Env Gp52 protein. All the antibodies detected MMTV proteins with exception of two out of four monoclonal antibodies against HMTV Env. Approximately 13% of the MSSM cells showed HMTV protein expression by FACS analysis. This report shows the expression of HMTV proteins for the first time in human breast cancer cells using a panel of antibodies against HMTV, HBRV and MMTV proteins. This should be taken into consideration when MMTV antibodies are used to detect HMTV proteins in human tissues. © 2009 Elsevier B.V. All rights reserved. Fil: Melana, Stella M.. Mount Sinai School of Medicine; Estados Unidos Fil: Nepomnaschy, Irene. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Mount Sinai School of Medicine; Estados Unidos Fil: Hasa, Jennifer. Mount Sinai School of Medicine; Estados Unidos Fil: Djougarian, Alina. Mount Sinai School of Medicine; Estados Unidos Fil: Djougarian, Anna. Mount Sinai School of Medicine; Estados Unidos Fil: Holland, James F.. Mount Sinai School of Medicine; Estados Unidos Fil: Pogo, Beatriz G.T.. Mount Sinai School of Medicine; Estados Unidos

Earlier studies have suggested an association between breast cancer aggressiveness and the presence of viral sequences resembling mouse mammary tumor virus (MMTV). This study was to determine if inflammatory breast cancer (IBC) in the United States had a higher proportion of cases with these viral sequences than non-IBC patients and if specific risk factors for the sequences could be identified. Biospecimens from 67 patients in the North American IBC Registry were selected for sequencing of MMTV env- like sequences. The presence or absence of the viral sequences was compared to progression free survival (PFS), risk factors including exogenous hormones, and tumor markers. Of the 67 cases, 44 were positive for viral sequences (VSP), 17 were negative (VSN) and six were excluded from analysis because of insufficient DNA to perform replicates. The 72% of VSP cases was significantly more than the 40% in non-IBC U.S. breast cancer patients (p < 0.0001). Non-significant trends suggested that VSP patients were more likely to be HER-2 neu positive and ER negative, have a stronger exposure to exogenous hormones, and have a shorter PFS than VSN patients. MMTV-related sequences appear to be related to the aggressiveness of breast cancer with a higher incidence in North American IBC than in non-IBC breast cancer.

In a single-institution retrospective cohort study, 230 patients were treated for stage III primary ovarian cancer and 175 became eligible for second-look operations by virtue of a complete clinical response after primary surgical cytoreduction and platinum-based combination chemotherapy. Of these, 109 underwent a second-look operation. Optimal primary cytoreduction was defined as residual disease ≤1 cm. Median follow-up was 68.3 months. Five-year survival for all the 230 stage III ovarian cancers was 43.4%. Among all eligible patients (n = 175), there was no survival difference (P = 0.67) in those having second look (57.3%, 5-year survival) versus no second look (48.7%). In those patients with optimal primary cytoreduction (n = 118), there was no survival advantage to second look (69% versus 61%, P = 0.7). However, in those with suboptimal primary cytoreduction (n = 47), 5-year survival was 36% in those having second look versus only 13% in those refusing second look (P < 0.05). Multivariate analysis identified second-look surgery as the only significant independent prognostic variable affecting survival (RR = 0.321, P < 0.04). Patients with suboptimal debulking at primary surgery for stage III ovarian cancer appear to achieve a survival benefit from second-look surgical procedures, presumably from the early identification and treatment of residual disease.

Prostate androgen regulated (PAR) is a 1038 bp novel gene located on chromosome 1 in epidermal differentiation complex. The gene is ubiquitously expressed in normal tissues and is overexpressed in most of their malignant counterparts. PAR cellular function is unknown. Here we report the effect of increased PAR expression induced by transfection of PAR cDNA on NIH3T3 cell phenotype. PAR-NIH3T3 transfectants expressing 3- to 4-fold higher PAR levels compared to controls grew faster in tissue cultures, formed colonies in soft agar, and exhibited a shortening of G1 and S phases of cell cycle and formed tumors in SCID mice. Transfection of NIH3T3 cells with increased ectopic PAR expression with a 22 mer oligonucleotide in antisense orientation with PAR mRNA abrogated their ability to form colonies in soft agar. The data presented here along with our previously reported results on DU145 cells transfected with antisense PAR cDNA suggest that PAR gene behaves like a proto-oncogene.

Purpose: Using a 2 × 2 factorial design, we studied the adjuvant chemotherapy of women with axillary node–positive breast cancer to compare sequential doxorubicin (A), paclitaxel (T), and cyclophosphamide (C) with concurrent doxorubicin and cyclophosphamide (AC) followed by paclitaxel (T) for disease-free (DFS) and overall survival (OS); to determine whether the dose density of the agents improves DFS and OS; and to compare toxicities. Patients and Methods: A total of 2,005 female patients were randomly assigned to receive one of the following regimens: (I) sequential A × 4 (doses) → T × 4 → C × 4 with doses every 3 weeks, (II) sequential A × 4 → T × 4 → C × 4 every 2 weeks with filgrastim, (III) concurrent AC × 4 → T × 4 every 3 weeks, or (IV) concurrent AC × 4 → T × 4 every 2 weeks with filgrastim. Results: A protocol-specified analysis was performed at a median follow-up of 36 months: 315 patients had experienced relapse or died, compared with 515 expected treatment failures. Dose-dense treatment improved the primary end point, DFS (risk ratio [RR] = 0.74; P = .010), and OS (RR = 0.69; P = .013). Four-year DFS was 82% for the dose-dense regimens and 75% for the others. There was no difference in either DFS or OS between the concurrent and sequential schedules. There was no interaction between density and sequence. Severe neutropenia was less frequent in patients who received the dose-dense regimens. Conclusion: Dose density improves clinical outcomes significantly, despite the lower than expected number of events at this time. Sequential chemotherapy is as effective as concurrent chemotherapy.

Gestational breast cancer (BC) is generally associated with rapid growth and increased mortality. Because the presence of MMTV-like sequences in BC has been associated with laminin receptor expression, a marker of poor prognosis, gestational BCs were analyzed for MMTV env gene-like sequences to explore whether MMTV-like sequences were also associated with its adverse outcome. Whereas 30-38% of sporadic BC have the sequences, in gestational BC the prevalence is 62%. We suggest that hormonal response elements present in the MMTV-like LTR may play a role in promoting cell growth, as they do in the mouse system.

Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E(2) (PGE(2)) restores hypoxic effects on VEGF. We hypothesized that PGE(2) mediates hypoxic effects on VEGF by modulating HIF-1alpha expression. Addition of PGE(2) to PC-3ML human prostate cancer cells had no effect on HIF-1alpha mRNA levels. However, PGE(2) significantly increased HIF-1alpha protein levels, particularly in the nucleus. This effect of PGE(2) largely results from the promotion of HIF-1alpha translocation from the cytosol to the nucleus. PGE(2) addition to PC-3 ML cells transfected with a GFP-HIF-1alpha vector induced a time-dependent nuclear accumulation of the HIF-1alpha protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1alpha levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE(2) reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE(2) effects on HIF-1alpha were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE(2) production via COX-2-catalyzed pathway plays a critical role in HIF-1alpha regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.

Arsenic trioxide (As(2)O(3)) has been found to induce apoptosis in leukemia cell lines and clinical remissions in patients with acute promyelocytic leukemia. In this study, we investigated the cytotoxic effect and mechanisms of action of As(2)O(3) in human tumor cell lines. As(2)O(3) caused inhibition of cell growth (IC(50) range, 3-14 microM) in a variety of human solid tumor cell lines, including four human non-small-cell lung cancer cell lines (H460, H322, H520, H661), two ovarian cancer cell lines (SK-OV-03, A2780), cervical cancer HeLa, and breast carcinoma MCF-7, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Flow cytometry analysis showed that As(2)O(3) treatment resulted in a time-dependent accumulation of cells in the G(2)/M phase. We observed, using Wright-Giemsa and 4',6-diamidine-2-phenylindole-dihydrochloride staining, that As(2)O(3) blocked the cell cycle in mitosis. In vitro examination revealed that As(2)O(3) markedly promoted tubulin polymerization without affecting GTP binding to beta-tubulin. Immunocytochemical and EM studies of treated MCF-7 cells showed that As(2)O(3) treatment caused changes in the cellular microtubule network and formation of polymerized microtubules. Similar to most anti-tubulin agents, As(2)O(3) treatment induced up-regulation of the cyclin B1 levels and activation of p34(cdc2)/cyclinB1 kinase, as well as Bcl-2 phosphorylation. Furthermore, activation of caspase-3 and -7 and cleavage of poly(ADP-ribose) polymerase and beta-catenin occurred only in As(2)O(3)-induced mitotic cells, not in interphase cells, suggesting that As(2)O(3)-induced mitotic arrest may be a requirement for the activation of apoptotic pathways. In addition, As(2)O(3) exhibited similar inhibitory effects against parental MCF-7, P-glycoprotein-overexpressing MCF-7/doxorubicin cells, and multidrug resistance protein (MRP)-expressing MCF-7/etoposide cells (resistance indices, 2.3 and 1.9, respectively). Similarly, As(2)O(3) had similar inhibitory effect against parental ovarian carcinoma A2780 cells and tubulin mutation paclitaxel-resistant cell lines PTx10 and PTx22 (resistance indices, 0.86 and 0.93, respectively), suggesting that its effect on tubulin polymerization and G(2)/M phase arrest is distinct from that of paclitaxel. Taken together, our data demonstrate that As(2)O(3) has a paclitaxel-like effect, markedly promotes tubulin polymerization, arrests cell cycle at mitosis, and induces apoptosis. In addition, As(2)O(3) is a poor substrate for transport by P-glycoprotein and MRP, and non-cross-resistant with paclitaxel resistant cell lines due to tubulin mutation, suggesting that As(2)O(3) may be useful for treatment of human solid tumors, particularly in patients with paclitaxel resistance.

PURPOSE: Patients with high-risk myelodysplastic syndrome (MDS) have high mortality from bone marrow failure or transformation to acute leukemia. Supportive care is standard therapy. We previously reported that azacitidine (Aza C) was active in patients with high-risk MDS. PATIENTS AND METHODS: A randomized controlled trial was undertaken in 191 patients with MDS to compare Aza C (75 mg/m2/d subcutaneously for 7 days every 28 days) with supportive care. MDS was defined by French-American-British criteria. New rigorous response criteria were applied. Both arms received transfusions and antibiotics as required. Patients in the supportive care arm whose disease worsened were permitted to cross over to Aza C. RESULTS: Responses occurred in 60% of patients on the Aza C arm (7% complete response, 16% partial response, 37% improved) compared with 5% (improved) receiving supportive care (P < .001). Median time to leukemic transformation or death was 21 months for Aza C versus 13 months for supportive care (P = .007). Transformation to acute myelogenous leukemia occurred as the first event in 15% of patients on the Aza C arm and in 38% receiving supportive care (P = .001). Eliminating the confounding effect of early cross-over to Aza C, a landmark analysis after 6 months showed median survival of an additional 18 months for Aza C and 11 months for supportive care (P = .03). Quality-of-life assessment found significant major advantages in physical function, symptoms, and psychological state for patients initially randomized to Aza C. CONCLUSION: Aza C treatment results in significantly higher response rates, improved quality of life, reduced risk of leukemic transformation, and improved survival compared with supportive care. Aza C provides a new treatment option that is superior to supportive care for patients with the MDS subtypes and specific entry criteria treated in this study.

Cyclooxygenase (COX)-2 expression and prostaglandin E(2) (PGE(2)) secretion are increased in prostatic intraepithelial neoplasia (PIN) and prostate cancer. PGE(2) biosynthesis by cyclooxygenase (COX)-2 plays a pivotal role in inflammation and carcinogenesis. One of the critical proinflammatory cytokines in the prostate is interleukin-6 (IL-6). We hypothesized that increased expression of COX-2, with resultant increased levels of PGE(2) in human PIN cells, activates the IL-6 signaling pathway. We demonstrate an autocrine upregulation of PGE(2) mediated by IL-6 in a human PIN cell line. We further demonstrate that PGE(2) stimulates soluble IL-6 receptor (sIL-6R) release, gp130 dimerization, Stat-3 protein phosphorylation, and DNA binding activity. These events, induced by PGE(2), lead to increased PIN cell growth. Treatment of PIN cells with a selective COX-2 inhibitor decreases cell growth. Finally, PGE(2)-stimulated PIN cell growth was abrogated by the addition of IL-6 neutralizing antibodies. These data provide mechanistic evidence that increased expression of COX-2/PGE(2) contributes to prostate cancer development and progression via activation of the IL-6 signaling pathway.

Human Mammary Tumor Virus (HMTV), a retrovirus 90-98% homologous to Mouse Mammary tumor Virus (MMTV), the etiological agent of murine mammary cancer, has been detected by PCR in 40% of American women’s breast cancers, but not in healthy tissues of the same breast (Cancer Research 1995, 55:5173-79; Clin. Cancer Research 2001, 7-283-4). A complete 9.9 kb HMTV proviral sequence has been detected in breast cancer genomic DNA (Cancer Res 2001,61:1754-9). HMTV viral particles with betaretrovirus characteristics have been isolated from metastatic breast cancer cells in effusions (MSSM cells). Expression of HMTV proteins has been detected in MSSM cells by western blot, fluorescence-activated cell sorting analysis, and immunofluorescence assays but not in normal mammary epithelial cells (J Virol Methods 2010, 163(1): 157-61). PCR is a sensitive technique susceptible of contamination, which due to amplification, could result in false positive detection. Although murine DNA was not found by PCR, the possibility still exists that HMTV in human DNA could be a result of laboratory contamination. Despite the substantial evidence supporting the presence of HMTV in breast cancer the controversy continues because the chance of contamination has led to doubt that murine-like viruses are human pathogens. We now report additional analyses of breast cancer specimens employing other methods for HMTV detection that reinforce previous findings. NanoString nCounter, a new technology using specific 100 mer oligonucleotides as probes bound to reporter molecules that can detect HMTV gag, env and LTR sequences as well as two murine specific probes in a single reaction without amplification. Flourescence in situ hybridization (FISH) assays with a HMTV probe is a technology that permits visualization of HMTV proviral DNA in the nuclei of surgical samples of breast cancer as well as in MSSM cells. Results from Nanostring nCounter, and FISH analysis show the presence of HMTV DNA in surgical cancer specimens and in MSSM cells. The data exclude contamination and confirm the authenticity of HMTV in human breast cancer. Next-generation sequencing and other studies are in progress to determine the role of HMTV in the pathogenesis of breast carcinoma. Citation Format: Stella M. Melana, Joseph Tripodi, Digna Nosike, Jaffer Shabnam, Polly Etkind, Beatriz GT Pogo, James F. Holland. Study of human mammary tumor virus (HMTV) in human breast cancer by NanoString nCounter and FISH analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5757. doi:10.1158/1538-7445.AM2017-5757

We have previously detected an MMTV env gene-like 660 bp sequence in 38% of human breast cancers, but not in normal tissues or other tumors. In this communication we report the sequences from eleven tumors and three breast cancer cell lines, and compare them to four strains of MMTV and to the known endogenous retroviral sequences. The breast cancer sequences were highly homogenous to the MMTV's, but not to the endogenous sequences suggesting an exogenous origin.

Background PAR is a novel gene ubiquitously expressed in normal and malignant tissues with a trend towards higher expression in tumor cells. PAR biological function is unknown. Here we report the effect of lowering PAR expression on in vitro and in vivo proliferation of DU145 cells. Methods Decreased PAR expression was achieved by stable transfection of DU145 cells with antisense PAR cDNA cloned in pCMV-Script expression vector. The proliferative potential of DU145 transfectants was studied by cell counts, colony formation in soft agar, flow cytometry, and growth in severe combined immunodeficient (SCID) mice. Results DU145 transfectants exhibited a decreased cell proliferation in tissue culture and a low efficiency of colony formation in soft agar. Flow cytometry revealed an arrest of these cells in G2-M phase of mitotic cycle. A dramatic decrease of tumor growth was observed when DU145 transfectant cells were inoculated in SCID mice, compared with controls. Histological examination of these tumors showed a marked decrease in cell density and in number of mitoses while control tumors showed a high cell density and numerous mitoses. Conclusions The data presented here provide the first evidence for PAR gene cellular function and its possible implication in malignant transformation. Prostate 49:200–207, 2001. © 2001 Wiley-Liss, Inc.

The mechanism underlying the cancericidal activity of 3-m-bromoacetylamino benzoic acid ethyl ester (3-BAABE) was investigated. 3-BAABE exerted a strong cancericidal effect on human leukemia and lymphoma cells (IC(50)0.2 microgram/mL) and on cell lines of prostate, colon, ductal, and kidney cancer (IC(50) 0.8 to 0.88 microgram/mL). Multiple drug resistance (MDR) had no effect on the susceptibility of human lymphoma cells to 3-BAABE, since Daudi/MDR(20) and wild-type Daudi cells had a similar susceptibility to the cytotoxic effect of 3-BAABE. The cancericidal effect of 3-BAABE, which was not associated with changes in the cell cycle, was mediated by apoptosis. Thus, cells exposed to 3-BAABE displayed the DNA fragmentation ladder characteristic for apoptosis, associated with a marked increase of the activity of apoptosis effector caspases-3 and -6, which was followed by proteolytic cleavage of DNA fragmentation factor (DFF) and poly(ADP-ribose) polymerase (PARP). Exposure of tumor cells to 3-BAABE increased the activity of apical caspase-9, but had no effect on caspase-8. Complete inhibition of 3-BAABE-induced apoptosis was exerted by LEHD-FMK, a caspase-9 inhibitor. DEVD-FMK, a caspase-3 inhibitor, and VEID-FMK, a caspase-6 inhibitor, partially inhibited 3-BAABE-induced apoptosis, whereas exposure to IETD-FMK, a caspase-8 inhibitor, had no effect. The fragmentation and elevated activity of caspase-9 in 3-BAABE-treated cells and the fact that only an inhibitor of caspase-9 abrogated 3-BAABE-induced apoptosis indicate that 3-BAABE is a distinctive compound that elicits apoptosis through a pathway that is limited specifically to activation of apical caspase-9.

Cyclooxygenase (COX)-2, an inducible enzyme which catalyzes the formation of prostaglandins from arachidonic acid, is expressed in prostate cancer specimens and cell lines. To evaluate the in vivo efficacy of a COX-2 inhibitor in prostate cancer, NS398 was administered to mice inoculated with the PC-3 human prostate cancer cell line.A total of 28 male nude mice were inoculated subcutaneously with 1 million PC-3 cells. Tumors were palpable in all 28 animals 1 week after inoculation and mice were randomized to receive either vehicle (control) or NS398, 3 mg./kg. body weight, intraperitoneally three times weekly for 9 weeks. Tumors were measured at weekly intervals. After a 10-week experimental period, mice were euthanized and tumors were immuno- histochemically assayed for proliferation (PCNA), apoptosis (TUNEL) and microvessel density (MVD) (Factor-VIII-related antigen). Tumor VEGF content was assayed by Western blotting.NS398 induced a sustained inhibition of PC-3 tumor cell growth and a regression of existing tumors. Average tumor surface area from control mice was 285 mm.2 as compared with 22 mm.2 from treated mice (93% inhibition, p0.001). Immunohistochemical analysis revealed that NS398 had no effect on proliferation (PCNA), but induced apoptosis (TUNEL) and decreased MVD (angiogenesis). VEGF expression was also significantly down regulated in the NS398-treated tumors.These results demonstrate that a selective COX-2 inhibitor suppresses PC-3 cell tumor growth in vivo. Tumor growth suppression is achieved by a combination of direct induction of tumor cell apoptosis and down regulation of tumor VEGF with decreased angiogenesis

Development of multidrug resistance (MDR) is the major obstacle to successful cancer chemotherapy. We have developed Daudi human lymphoma cells that are 20-fold more resistant than the parent cell line to vincristine (VCR) by infecting cells with pHaMDR1/A retroviral vector (Daudi/MDR20). Three DNA sequences of anti-MDR1 hammerhead ribozymes (Rzs), one cleaving codon 196 of MDR1 mRNA (196MDR1-Rz), the second a stem II base-modified (U9--Gg, U13--A13, G14--A14, A18--C18) Rz against codon 196 (196MDR1-sRz), and the third a stem II base-modified Rz directed against the -6 approximately -4 GUC sequence of the translation initiation site of the MDR1 mRNA (iMDR1-sRz), were synthesized and cloned into the retroviral vector N2A+tRNAiMet downstream of the RNA polymerase III promoter and adjacent to a tRNA gene sequence, forming the constructs N2A+tRNAiMet-196MDR1-Rz, N2A+tRNAiMet-196MDR1-sRz, and N2A+tRNAiMet-iMDR1-sRz. The three constructs were transfected into GP+envAM 12 cells for packaging the retroviral vectors. The supernatants containing the packaged retrovirus in high titers (1.1-2.5 X 10(5) CFU/ml as determined by infection of NIH 3T3 cells) were used to infect Daudi/MDR20 cells. The iMDR1-sRz- and 196MDR1-sRz-transduced Daudi/MDR20 cells completely restored chemosensitivity to VCR and doxorubicin, and were accompanied by blocked expression of MDR1 mRNA and P-glycoprotein as well as overexpression of anti-MDR1 Rz. In a cell-free system, the chimeric tRNA-sRz molecules were more stable and had more efficient catalytic activities than the corresponding naked Rz molecules. The stem II base-modified Rz were also more stable and efficient in catalytic activities than the unmodified Rz molecules. The base modification in the Rz stem II structure and the development of chimeric tRNA-Rz molecules were identified to enhance the cleavage efficacy. The combination of these two factors, together with the use of a retroviral vector, appear to have contributed to the complete reversal of MDR.

Upregulation of vascular endothelial growth factor (VEGF) expression induced by hypoxia is crucial event leading to neovascularization. Cyclooxygenase-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, has been demonstrated to be induced by hypoxia and play role in angiogenesis and metastasis. To investigate the potential effect of COX-2 on hypoxia-induced VEGF expression in prostate cancer. We examined the relationship between COX-2 expression and VEGF induction in response to cobalt chloride (CoCl2)-simulated hypoxia in three human prostate cancer cell lines with differing biological phenotypes. Northern blotting and ELISA revealed that all three tested cell lines constitutively expressed VEGF mRNA, and secreted VEGF protein to different degrees (LNCaP > PC-3 > PC3ML). However, these cell lines differed in the ability to produce VEGF in the presence of CoCl2-simulated hypoxia. CoCl2 treatment resulted in 40% and 75% increases in VEGF mRNA, and 50% and 95% in protein secretion by LNCaP and PC-3 cell lines, respectively. In contrast, PC-3ML cell line, a PC-3 subline with highly invasive, metastatic phenotype, exhibits a dramatic upregulation of VEGF, 5.6-fold in mRNA and 6.3-fold in protein secretion after treatment with CoCl2. The upregulation of VEGF in PC-3ML cells is accompanied by a persistent induction of COX-2 mRNA (6.5-fold) and protein (5-fold). Whereas COX-2 expression is only transiently induced in PC-3 cells and not affected by CoCl2 in LNCaP cells. Moreover, the increases in VEGF mRNA and protein secretion induced by CoCl2 in PC-3ML cells were significantly suppressed following exposure to NS398, a selective COX-2 inhibitor. Finally, the effect of COX-2 inhibition on CoCl2-induced VEGF production was reversed by the treatment with exogenous PGE2. Our data demonstrate that VEGF induction by cobalt chloride-simulated hypoxia is maintained by a concomitant, persistent induction of COX-2 expression and sustained elevation of PGE2 synthesis in a human metastatic prostate cancer cell line, and suggest that COX-2 activity, reflected by PGE2 production, is involved in hypoxia-induced VEGF expression, and thus, modulates prostatic tumor angiogenesis.

Androgens are known to directly stimulate prostate cancer cell growth. We have previously reported that LNCaP prostate cancer cells were dependent upon stromal coinoculation for growth in nude mice and that the stromal cells secreted a potent angiogenic factor, vascular endothelial growth factor (VEGF), which stimulated tumor angiogenesis. Immunohistochemical staining localized VEGF expression primarily to the stromal cells of human fetal and adult hyperplastic prostates, with both stromal and epithelial cell VEGF expression in prostate cancer. In the present studies, we test the hypothesis that androgens, in addition to their direct effects on prostate epithelial cells, have indirect effects on these cells via up-regulation of stromal VEGF production and angiogenesis. Primary cultures of human prostate fetal fibroblasts were treated with dihydrotestosterone (DHT), and the effects on VEGF messenger RNA (mRNA) expression were determined by Northern blotting. DHT (10 nm) increased VEGF mRNA levels maximally...

Suramin, an organic polyanion with six sulphonic groups, is under clinical trials as an agent against hormone-refractory prostate cancer. The drug binds strongly to serum albumin. The objectives were to use electrospray to measure the molecular masses of the intact complexes of albumin and suramin to determine the number of suramin molecules bound under different molar ratios, and to investigate the binding of suramin in human serum. With albumin in excess (2:1 to 25:1 ratio), only 1 and 2 bound suramins were found; with suramin excess (2:1 to 1000:1) up to 20 bound suramin molecules/albumin were found. Up to 5 bound suramins were found in human serum with 4:1 suramin:albumin ratio, which corresponds to recommended therapeutic doses (200–300 μg/mL). At 8:1 ratio, which would be toxic, complexes with up to ten bound suramin molecules were found, and unreacted albumin diminished. © 1998 John Wiley & Sons, Ltd.

Purpose: Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. We examined whether cationic liposome-mediated transfer of a ribozyme could reverse MDR. Methods: A ribozyme which cleaved codon 196 of MDR1 mRNA was constructed from synthetic oligonucleotides. The MDR1 ribozyme was mixed with N-(1-(2,3-dileoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (DOTAP) to form a liposomal complex. The complex was used to treat two P-glycoprotein-producing MDR cell lines: MCF-7/R human breast cancer cells resistant to doxorubicin and MOLT-3/TMQ800 human ALL cells resistant to trimetrexate (TMQ). In order to investigate the differential sensitivity of these two cell lines to the liposome-ribozyme complex, cellular pharmacological studies including phase-contrast and confocal microscopic studies were performed. Results: Treatment with the liposome-ribozyme complex resulted in reversal of vincristine (VCR) resistance in MCF-7/R cells, but not in MOLT-3/TMQ800 cells. In MCF-7/R cells the treatment resulted in decreases in MDR1 mRNA expression and P-glycoprotein production, whereas no changes in these parameters were seen in MOLT-3/TMQ800 cells. Phase-contrast microscopy revealed that in MCF-7/R cells treatment with DOTAP led to the formation of cytoplasmic vacuoles, and treatment with latex beads resulted in the development of a shiny material in the cytoplasm. In contrast, in MOLT-3/TMQ800 cells hardly any morphological changes occurred. Confocal microscopic imaging showed cytoplasmic fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads. In MOLT-3/TMQ800 cells no fluorescence was detected. Treatment with cytochalasin B abolished fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads. These studies show that MCF-7/R cells have high endocytotic activity whereas MOLT-3/TMQ800 cells have little activity. Conclusions: Endocytotic activity was correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme. Determination of endocytotic activity of target tumor cells may be predictive of efficacy of liposome-mediated gene transfer.

Vincristine (VCR) is an active agent in the treatment of acute lymphoblastic leukemia (ALL). We evaluated the relationship between the cytotoxic activity of VCR and the degree of VCR saturation of cellular drug binding sites, using the MOLT-3 ALL cell line. When MOLT-3 cells at a density of 1 × 10 6 or 1 × 10 8 cells/mL of pH-controlled medium were exposed to VCR for 1 hr, its cytotoxic activity on cells at high density was 10-fold less than on cells at low density (inoculum effect). The number of VCR binding sites measured by Scatchard analysis was 9.25 × 10 6 /cell. At high cell density, the saturation of VCR binding sites was one log order less than that at low density. Irrespective of cell density, curves of cell-kill versus the degree of VCR saturation of the cellular binding sites overlapped each other. Minimal cytotoxic activity was observed at 0.3% VCR saturation, and nearly maximal cytotoxic activity occurred at about 25% saturation, with the ic 50 at about 4% saturation. These data show that the VCR-induced cell-kill effect is dependent on the degree of saturation of VCR binding sites rather than on the extracellular VCR concentration. The lesser cell-kill on cells at high density can be explained by the lack of drug molecules to sufficiently saturate cellular binding sites. This phenomenon may be responsible, at least in part, for the poor chemotherapeutic outcome of ALL patients with high leukocyte counts at presentation.

Background and Objectives: Malignant mesothelioma has a poor prognosis and is refractory to many agents. The antitumor effectiveness of cisplatin, paclitaxel, and suramin as single agents and in combination was evaluated in vivo against four lines of human pleural malignant mesothelioma xenografts in athymic nude mice, including one epithelial type and three fibrosarcomatous. Methods After growth of tumors occurred by day 54 or 55, mice were randomized in groups of four each to receive either cisplatin 4 mg/kg intraperitoneally weekly ×5, or paclitaxel (Taxol) 12.5 mg/kg subcutaneously daily 5 days/week for 3 consecutive weeks, or suramin 60 mg/kg intraperitoneally daily ×4,versus controls treated with normal saline. Results Cisplatin was very effective against one line and also to a lesser degree against another line. Paclitaxel showed antitumor effects similar to cisplatin, being very effective in one line, and also showed good activity in another line. Suramin was basically inactive in all four lines. Following the results obtained with these single agents, it was decided to evaluate the combination of cisplatin and paclitaxel, which resulted in more pronounced antitumor effect in all four cell lines. Conclusions These results indicate that the combination of cisplatin and paclitaxel is superior to each agent alone in this model, and that it deserves to be evaluated in patients with malignant mesothelioma. J. Surg. Oncol. 1998;67:104–111. © 1998 Wiley-Liss, Inc.

BACKGROUND Although previous research has delineated medical, cognitive, and neuropsychologic late effects of central nervous system (CNS) prophylaxis for childhood acute lymphoblastic leukemia (ALL), it has been difficult to draw conclusions about the long term psychosocial sequelae of these treatments due to methodologic problems that led to inconclusive results in past studies. In the current study, the authors examined the long term psychosocial functioning of childhood ALL survivors who had been treated on a Phase III clinical protocol (Cancer and Leukemia Group B [CALGB] 7611) between 1976 and 1979, in which they were randomized to receive either 2400 centigray of cranial radiation (CRT) with intrathecal methotrexate (IT-MTX) or intermediate dose systemic methotrexate (IV-MTX) with IT-MTX. METHODS One hundred ten survivors of childhood ALL (mean age, 20.8 years) treated on CALGB 7611 who were age 14 years or older and disease free for at least 1 year were studied a mean of 14.7 years after their entry on CALGB 7611. In a telephone interview, a psychosocial assessment battery was administered to the patients, consisting of measures that assessed psychologic, sexual, social, and vocational functioning as well as any delayed physical effects. RESULTS Survivors who had received CRT + IT-MTX had significantly poorer academic achievement (P = 0.0001), poorer self-images with regard to their bodies (P = 0.001), and greater psychologic distress (P = 0.005). CONCLUSIONS Cranial radiation used to treat children with ALL has significant long term sequelae in terms of poorer academic achievement and psychosocial functioning. These data add weight to the conclusion that CRT prophylaxis should only be used to treat children who are at high risk of CNS relapse. Cancer 1998;82:208-18. © 1998 American Cancer Society.