Multicentric hepatic EBV-associated smooth muscle tumors in an AIDS patient: a case report, investigation of mTOR activation and review of the literature.

Epstein-Barr virus (EBV) - associated smooth muscle tumors (EBV-SMT) are a rare, recently recognized distinct group of mesenchymal tumors that develop exclusively in patients with immunosuppression. It is believed that tumorigenesis is, at least in part, through the activation of the Akt/mammalian target of rapamycin (mTOR) signal pathway. We describe the clinicopathologic and immunohistochemical features of a multifocal hepatic EBV-SMT in a 34-year-old acquired immunodeficiency syndrome (AIDS) patient and investigate the activation status of the mTOR signal pathway in this tumor. In addition, we provide a review of the literature on the clinicopathologic findings of hepatic EBV-SMT in adult AIDS patients, and discuss their biologies and possible therapeutic strategies.

Tumors in the contralateral breast following radiation therapy for breast cancer

The proportional distribution of independent malignant tumors in the contralateral breast following treatment for breast cancer was investigated to assess the influence of scattered radiation as a cause of these tumors. In a population of 172 patients the proportion of contralateral tumors in each quadrant and the center (the nipple-areolar complex) was compared with the expected, or natural, distribution found in the general population, in the absence of radiation. The observed/expected ratio for contralateral tumors was 1.43 for the upper-inner quadrant; 0.97, lower-inner quadrant; 1.51, center; 0.76, upper-outer quadrant; and 0.64, lower-outer quadrant. In each quadrant, except the lower-inner, the observed/expected ratio differed from 1.00 with statistical significance at the 5% level (one-tail). The same analysis, stratified by age and menopausal status, showed a similar shift of tumors, with more than expected in the inner quadrants and center and less than expected in the outer quadrants, although the results did not show statistical significance at the 5% level for all strata. For each patient the mean absorbed radiation dose for each quadrant and center of the breast was estimated, based on measurements in a tissue-equivalent phantom. Among patients the doses ranged from 0.5 to 8 Gy; within individuals, doses to the inner quadrants typically were a factor of three times higher than doses to the outer quadrants. The results suggest that radiation may be a risk factor for contralateral breast tumors and warrants further investigation. ^

AN EPIDEMIOLOGIC CASE-CONTROL ANALYSIS OF CHILDHOOD INTRACRANIAL AND SPINAL CORD TUMORS IN RELATION TO PATERNAL OCCUPATION AT BIRTH (BRAIN, CANCER)

Epidemiologic case-control studies of small groups of childhood nervous system tumor patients have suggested that parental employment in occupations with exposure to hydrocarbons is a risk factor for disease. The main focus of this case-control study was to assess the paternal occupation at the time of birth of offspring who later developed childhood intracranial and spinal tumors. All children under 15 years of age dying of such tumors in Texas, during the period 1964-1980, were selected as cases. Disease and demographic data were abstracted from death certificates. The birth certificate for each child of the final group of 499 cases was located and parental occupation information, as well as demographic and obstetric data, were collected. The comparison group consisted of a random sample from all Texas live births with the same birth year, race and sex distribution as the cases.^ The paternal occupations were categorized into broad classifications of those involving hydrocarbon exposure versus those that did not, based on the occupation criteria used in the previous studies. Odds ratios did not indicate any increased risk associated with general paternal hydrocarbon exposure in the workplace. In prior studies, increased risk estimates were detected with narrower groups of occupations involving exposure to hydrocarbon materials. The data from this study were classified according to these groups, and again, no increased risks were indicated except for a statistically insignificant but elevated odds ratio for fathers who were paper and pulp mill workers.^ Odds ratios were calculated for specific occupations and industries previously implicated as risk factors. Significantly associated odds ratios (OR) were detected for electricians (OR = 3.5), especially those working for construction companies (OR = 10.0), for employment in the printing occupations (OR = 4.5), particularly graphic arts workers (OR = 21.9), and in the electronics and electronic machinery industries (OR = 3.5). Analysis of the petroleum refining and chemical industries, which were not found in previous study populations, revealed significantly elevated odds ratios of 3.0 for occupations with probable heavy exposure to chemicals and petroleum compounds and 10.0 for salesmen of chemical products. ^

Abnormal expression of REST/NRSF and Myc in neural stem/progenitor cells causes cerebellar tumors by blocking neuronal differentiation.

Medulloblastoma, one of the most malignant brain tumors in children, is thought to arise from undifferentiated neural stem/progenitor cells (NSCs) present in the external granule layer of the cerebellum. However, the mechanism of tumorigenesis remains unknown for the majority of medulloblastomas. In this study, we found that many human medulloblastomas express significantly elevated levels of both myc oncogenes, regulators of neural progenitor proliferation, and REST/NRSF, a transcriptional repressor of neuronal differentiation genes. Previous studies have shown that neither c-Myc nor REST/NRSF alone could cause tumor formation. To determine whether c-Myc and REST/NRSF act together to cause medulloblastomas, we used a previously established cell line derived from external granule layer stem cells transduced with activated c-myc (NSC-M). These immortalized NSCs were able to differentiate into neurons in vitro. In contrast, when the cells were engineered to express a doxycycline-regulated REST/NRSF transgene (NSC-M-R), they no longer underwent terminal neuronal differentiation in vitro. When injected into intracranial locations in mice, the NSC-M cells did not form tumors either in the cerebellum or in the cerebral cortex. In contrast, the NSC-M-R cells did produce tumors in the cerebellum, the site of human medulloblastoma formation, but not when injected into the cerebral cortex. Furthermore, the NSC-M-R tumors were blocked from terminal neuronal differentiation. In addition, countering REST/NRSF function blocked the tumorigenic potential of NSC-M-R cells. To our knowledge, this is the first study in which abnormal expression of a sequence-specific DNA-binding transcriptional repressor has been shown to contribute directly to brain tumor formation. Our findings indicate that abnormal expression of REST/NRSF and Myc in NSCs causes cerebellum-specific tumors by blocking neuronal differentiation and thus maintaining the "stemness" of these cells. Furthermore, these results suggest that such a mechanism plays a role in the formation of human medulloblastoma.

Prognostic factors in desmoid tumors

Purpose. To evaluate the prognostic factors in desmoid tumors in the light of its possible use in standardizing the treatment strategy of an individual patient. ^ Patients and methods. A retrospective review of 189 consecutive patients who were treated at MD Anderson Cancer Center (MDACC) from January 1995 to December 2005 was done. Univariate and multivariate analysis of different prognostic factors was done on all patients, patients treated with surgery alone, subset of patients who came to MDACC with primary tumor. The median follow up was 63 months. Also the analysis of 189 desmoid patients treated at MDACC between 1995 and 2005 was compared to results of 189 desmoid patients treated at MDACC from 1965-1994 using data retrieved from a 150 field prospective relational soft tissue tumor database. ^ Results. 5-, and 10-year overall survival rate were 0.976 (95%CI 0.952, 0.999), and 0.966 (95% CI 0.935, 0.996), respectively. 5-, and 10-year recurrence free rate were 0.803 (95%CI 0.738, 0.868), and 0.793 (95% CI 0.726, 0.860), respectively. 5 year recurrence free survival for surgery alone, radiotherapy alone, chemotherapy alone and combination regimen were 0.759, 0.625, 0.933, and 0.802 respectively. Age (>30 vs. <=30) and primary tumor site (extremity vs visceral) were two prognostic factors significantly associated with local recurrence in all of the patients. ^ Conclusion. An increased awareness of the complex multidisciplinary management needed for successful control of desmoid tumor may underlie a significantly increased number of desmoid referrals, especially primary untreated desmoids, to UTMDACC. The careful prospective integration of multiple therapies has led to a significant recent improvement in desmoid patient outcome. These trends should be supported, particularly if personalized molecular-based therapies are to be rapidly and effectively deployed for the benefit of those afflicted by this rare and potentially devastating disease.^

The role of p53 in skin cancer induction by UV radiation and as a potential tumor antigen in UV-induced murine skin tumors

Carcinoma of the skin is the most common type of human cancer in the United States. Ultraviolet radiation (UVR) present in the sunlight is thought to be the major carcinogen responsible for induction of skin cancer. In UV-associated skin carcinogenesis, mutations in p53 are not only present with very high frequency, but occur early in the course of tumor development. In addition, UV-induced skin tumors in mice exhibit unique immunological characteristics. They are highly antigenic and express both individually-specific tumor transplantation antigens recognized by effector T cells and the UV-associated common antigen recognized by UV-induced suppressor T cells. ^ To examine the hypothesis that p53 plays a critical role in preventing skin cancer induction by UVR, mice constitutively lacking one or two functional p53 alleles were compared to wild-type mice for their susceptibility to UV carcinogenesis. Both p53 +/– and –/– mice showed greater susceptibility to skin cancer induction than wild-type mice, and –/– mice were the most susceptible, Accelerated tumor development in the p53 +/– mice was not associated with loss of the remaining wild-type allele of p53 , but in many cases was associated with UV-induced mutations in p53. Our studies clearly demonstrate the essential role of p53 in protection against UV carcinogenesis, particularly in the eye and epidermis. ^ The role of p53 in the antigenicity of UV-induced murine skin tumors was also addressed. Primary UV-induced tumors from p53 –/–, +/– and +/+ mice were transplanted into both normal and immunosuppressed mice, and rates of tumor rejection were compared. Tumors from mice with only one or no functional p53 alleles were less antigenic than those from mice with two functional p53 alleles. Moreover, tumors with no functional p53 also failed to grow well in chronically UV-irradiated mice. These results indicate that p53 contributes to the strong antigenicity of UV-induced murine skin tumors, and suggest that it may play a critical role in expression of the UV-associated common antigen recognized by suppressor T cells. ^ In this study we also monitored the effect of UVR on the development of lymphoid malignancies in p53 deficient mice. The incidence of lymphoid malignancies in UV-irradiated p53 +/– mice was drastically enhanced compared to that in unirradiated counterparts. The immune responses of the mice were identical and were suppressed to the same extent by UV irradiation regardless of the p53 genotype. These data provide the first experimental evidence that exposure to UVR can contribute to the development of lymphoid neoplasms in genetically susceptible hosts. ^

Understanding acquired resistance to Lapatinib in breast cancer cells

Signaling through epidermal growth factor receptor (EGFR/ErbB) family members plays a very important role in regulating proliferation, development, and malignant transformation of mammary epithelial cells. ErbB family members are often over-expressed in human breast carcinomas. Lapatinib is an ErbB1 and ErbB2 tyrosine kinase inhibitor that has been shown to have anti-proliferative effects in breast and lung cancer cells. Cells treated with Lapatinib undergo G1 phase arrest, followed by apoptosis. Lapatinib has been approved for clinical use, though patients have developed resistance to the drug, as seen previously with other EGFR inhibitors. Moreover, the therapeutic efficacy varies significantly within the patient population, and the mechanism of drug sensitivity is not fully understood. Expression levels of ErbB2 are used as a prognostic marker for Lapatinib response; however, even among breast tumor cell lines that express similar levels of ErbB2 there is marked difference in their proliferative responses to Lapatinib. To understand the mechanisms of acquired resistance, we established a cell line SkBr3-R that is resistant to Lapatinib, from a Lapatinib-sensitive breast tumor cell line, SkBr3. We have characterized the cell lines and demonstrated that Lapatinib resistance in our system is not facilitated by receptor-level activity or by previously known mutations in the ErbB receptors. Significant changes were observed in cell proliferation, cell migration, cell cycle and cell death between the Lapatinib resistant SkBr3-R and sensitive SkBr3 cell lines. Recent studies have suggested STAT3 is upregulated in Lapatinib resistant tumors in association with ErbB signaling. We investigated the role that STAT3 may play in Lapatinib resistance and discovered higher STAT3 activity in these resistant cells. In addition, transcriptional profiling indicated higher expression of STAT3 target genes, as well as of other genes that promote survival. The gene array data also revealed cell cycle regulators and cell adhesion/junction component genes as possible mediator of Lapatinib resistance. Altogether, this study has identified several possible mechanisms of Lapatinib resistance.

APPLICATIONS OF EPHB4 RECEPTOR SPECIFIC PEPTIDES IN TARGETED CANCER IMAGING AND THERAPY

EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are found over-expressed in a variety of tumors cells including glioma cells as well as angiogenic blood vessels. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. Targeted delivery of EphB4 receptor specific peptide conjugated hollow gold nanoshells (HAuNS) into tumors has great potential in cancer imaging and photothermal therapy. In this study, we developed an EphB4 specific peptide named TNYL-RAW and labeled with radioisotope 64Cu and Cy5.5 dye. We also conjugate this specific peptide with hollow gold nanoshells (HAuNS) to evaluate targeted photothermal therapy of cancers. In vitro, 64Cu-DOTA-TNYL- RAW specifically bind to CT26 and PC-3M cells but not to A549 cells. In vivo, Small-animal PET/CT clearly showed the significant uptake of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, µPET/CT and near-infrared optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice. In U251 tumors, Cy5.5-labeled peptide can bind to EphB4-expressing tumor blood vessels and tumors cells. But in U87 models, dual labeled peptide only could bind to tumor associated blood vessels. Also, Irradiation of PC-3M and CT-26 cell treated with TNYL-PEG-HAuNS nanopatilces with near-infrared (NIR) laser resulted in selective destruction of these cells in vitro. EphB4 targeted TNYL-PEG-HAuNS showed more photothermal killing effect on CT26 tumor model than PEG-HAuNS did. In summary, tumors with overexpression of EphB4 receptors can be noninvasively visualized by micro PET/CT with 64Cu labeled or dual labeled TNYL-RAW peptide. Targeted delivery of TNYL-RAW conjugated HAuNS into tumors can greatly improve the treatment effect of photothermal therapy. The information acquired with this study should be advantageous in improving diagnostics and future applications in photothermal ablation therapy in clinical.

THE ROLE OF TAK1 IN PANCREATIC CANCER DEVELOPMENT

Pancreatic cancer is the fourth leading cause of cancer-related mortality in the United States and the fifth leading cause of cancer-related mortality worldwide. Pancreatic cancer is a big challenge in large due to the lack of early symptoms. In addition, drug resistance is a major obstacle to the success of chemotherapy in pancreatic cancer. The underlying mechanism of drug resistance in human pancreatic cancers is not well understood. Better understanding of the mechanism of molecular pathways in human pancreatic cancers can help to identify the novel therapeutic target candidates, and develop the new preventive and clinic strategies to improve patient survival. We discovered that TAK1 is overexpressed in pancreatic cancer cell lines and patient tumor tissues. We demonstrated that the elevated activity of TAK1 is caused by its binding partner TAB1. Knocking down of TAK1 in pancreatic cancer cells with RNAi technique resulted in cell apoptosis and significantly reduces the size of tumors in mice and made a chemotherapy drug more potent. Targeting the kinase activity of TAK1 with the selective inhibitor LY2610956 strongly synergized in vitro with the antitumor activity of gemcitabine, oxaliplatin, or irinotecan on pancreatic cancer cells. These findings highlighted that TAK1 could be a potential therapeutic target for pancreatic cancer. We also demonstrated that TAK activity is regulated by its binding protein TAB1. We defined a minimum TAB1 sequence which is required and sufficient for TAK1 kinase activity. We created a recombinant TAK1-TAB1 C68 fusion form which has highly kinase activity. This active form could is used for screening TAK1 inhibitors. In addition, several posttranslational modifications were identified in our study. The acetylation of lysine 158 on TAK1 is required for kinase activity. This site is conserved throughout all of kinase. Our findings may reveal a new mechanism by which kinase activity is regulated.

UNDERSTANDING NANOG'S ROLE IN CANCER BIOLOGY

Understanding Nanog’s Role in Cancer Biology Mark Daniel Badeaux Supervisory Professor Dean Tang, PhD The cancer stem cell model holds that tumor heterogeneity and population-level immortality are driven by a subset of cells within the tumor, termed cancer stem cells. Like embryonic or somatic stem cells, cancer stem cells are believed to possess self-renewal capacity and the ability to give rise to a multitude of varieties of daughter cell. Because of cancer’s implied connections to authentic stem cells, we screened a variety of prostate cancer cell lines and primary tumors in order to determine if any notable ‘stemness’ genes were expressed in malignant growths. We found a promising lead in Nanog, a central figure in maintaining embryonic stem cell pluripotency, and through a variety of experiments in which we diminished Nanog expression, found that it may play a significant role in prostate cancer development. We then created a transgenic mouse model in which we targeted Nanog expression to keratin 14-expressing in order to assess its potential contribution to tumorigenesis. We found a variety of developmental abnormalities and altered differentiation patterns in our model , but much to our chagrin we observed neither spontaneous tumor formation nor premalignant changes in these mice, but instead surprisingly found that high levels of Nanog expression inhibited tumor formation in a two-stage skin carcinogenesis model. We also noted a depletion of skin stem cell populations, which underlies the wound-healing defect our mice harbor as well. Gene expression analysis shows a reduction in c-Jun and Bmp5, two genes whose loss inhibits skin tumor development and reduces stem cell counts respectively. As we further explored Nanog’s activity in prostate cancer, it became apparent that the protein oftentimes was not expressed. Emboldened by the competing endogenous RNA (ceRNA) hypothesis, we identified the Nanog 3’UTR as a regulator of the tumor suppressive microRNA 128a (miR-128a), which includes known oncogenes such as Bmi1 among its authentic targets. Future work will necessarily involve discerning instances in which Nanog mRNA is the biologically relevant molecule, as well as identifying additional mRNA species which may serve solely as a molecular sink for miR-128a.

Role of germ line p53 mutations in aneuploidy and immortalization of dermal fibroblasts from Li-Fraumeni cancer patients

Pedigree analysis of certain families with a high incidence of tumors suggests a genetic predisposition to cancer. Li and Fraumeni described a familial cancer syndrome that is characterized by multiple primary tumors, early age of onset, and marked variation in tumor type. Williams and Strong (1) demonstrated that at least 7% of childhood soft tissue sarcoma patients had family histories that is readily explained by a highly penetrant autosomal dominant gene. To characterize the mechanism for genetic predisposition to many tumor types in these families, we have studied genetic alterations in fibroblasts, a target tissue from patients with the Li-Fraumeni Syndrome (LFS).^ We have observed spontaneous changes in initially normal dermal fibroblasts from LFS patients as they are cultured in vitro. The cells acquire an altered morphology, chromosomal anomalies, and anchorage-independent growth. This aberrant behavior of fibroblasts from LFS patients had never been observed in fibroblasts from normal donors. In addition to these phenotypic alterations, patient fibroblasts spontaneously immortalize by 50 population doublings (pd) in culture; unlike controls that remain normal and senesce by 30-35 (2). At 50 pd, immortal fibroblasts from two patients were found to be susceptible to tumorigenic transformation by an activated T24 H-ras oncogene (3). Approximately 80% of the oncogene expressing transfectants were capable of forming tumors in nude mice within 2-3 weeks. p53 has been previously associated with immortalization of cells in culture and cooperation with ras in transfection assays. Therefore, patients' fibroblast and lymphocyte derived DNA was tested for point mutations in p53. It was shown that LFS patients inherited certain point mutations in one of the two p53 alleles (4). Further studies on the above LFS immortal fibroblasts have demonstrated loss of the remaining p53 allele concomitant with escape from senescence. While the loss of the second allele correlates with immortalization it is not sufficient to transformation by an activated H-ras or N-ras oncogene. These immortal fibroblasts are resistant to tumorigenic transformation by v-abl, v-src, c-neu or v-mos oncogene; implying that additional steps are required in the tumorigenic progression of LFS patients' fibroblasts.^ References. (1) Williams et al., J. Natl. Cancer Inst. 79:1213, 1987. (2) Bischoff et al., Cancer Res. 50:7979, 1990. (3) Bischoff et al., Oncogene 6:183, 1991. (4) Malkin et al., Science 250:1233, 1990. ^

Molecular analysis of TNF sensitivity in normal and Ha-{\it ras\/} transformed murine fibroblasts

Tumor necrosis factor (TNF) is known to have antiproliferative effects on a wide variety of tumor cells but proliferative effects on normal cells. However, the molecular basis for such differences in the action of TNF are unknown. The overall objectives of my research are to investigate the role of oncogenes in TNF sensitivity and delineate some of the molecular mechanisms involved in TNF sensitivity and resistance. To accomplish these objectives, I transfected TNF-resistant C3H mouse embryo fibroblasts (10T1/2) with an activated Ha-ras oncogene and determined whether these cells exhibit altered sensitivity to TNF. The results indicated that 10T1/2 cells transfected with an activated Ha-ras oncogene (10T-EJ) not only produced tumors in nude mice but also exhibited extreme sensitivity to cytolysis by TNF. In contrast, 10T1/2 cells transfected with the pSV2-neo gene alone were resistant to the cytotoxic effects of TNF. I also found that TNF-induced cell death was mediated through apoptosis. The differential sensitivity of 10T1/2 and 10T-EJ cell lines to TNF was not due to differences in the number of TNF receptors on their cell surface. In addition, TNF-resistant revertants isolated from Ha-ras-transformed, TNF-sensitive cells still expressed the same amount of p21 as TNF-sensitive cells and were still tumorigenic, suggesting that Ha-ras-induced transformation and TNF sensitivity may follow different pathways. Interestingly, TNF-resistant but not sensitive cells expressed higher levels of bcl-2, c-myc, and manganese superoxide dismutase (MnSOD) mRNA following exposure to TNF. However, TNF treatment resulted in a marginal induction of p53 mRNA in both TNF-sensitive and resistant cells. Based on these results I can conclude that (i) Ha-ras oncogene induces both transformation and TNF sensitivity, (ii) TNF-induced cytotoxicity involves apoptosis, and (iii) TNF-induced upregulation of bcl-2, c-myc, and MnSOD genes is associated with TNF resistance in C3H mouse embryo fibroblasts. ^

{\it In vivo\/} induction of DNA changes in cervicovaginal epithelium by perinatal estrogen exposure

Epidemiological studies have associated estrogens with human neoplasm such as the endometrium, cervix, vagina, breast, and liver. Perinatal exposure to natural (17$\beta$-estradiol (17$\beta$-E$\sb2)\rbrack$ and synthetic (diethylstilbestrol (DES)) estrogens induces neoplastic changes in humans and rodents. Previous studies demonstrated that neonatal 17$\beta$-E$\sb2$ treatment increased the nuclear DNA content of mouse cervicovaginal epithelium that preceded histologically evident neoplasia. In order to determine whether this effect was specific to 17$\beta$-E$\sb2,$ associated with chromosomal changes, and relevant to the human, female BALB/c mice were treated neonatally with either 17$\alpha$-estradiol (17$\alpha$-E$\sb2)$ and 5$\beta$-dihydrotestosterone ($5\beta$-DHT), both inactive steroids in adult reproductive tissue, or 17$\beta$-E$\sb2.$ Ten-day-old mice received pellet implants of 17$\beta$-E$\sb2,$ 17$\alpha$-E$\sb2,$ $5\beta$-DHT, or cholesterol. Seventy-day-old cervicovaginal tracts were examined histologically and flow cytometrically. 17$\beta$-E$\sb2$-treated animals were evaluated by fluorescent in situ hybridization (FISH) using a probe specific for chromosome 1. Trisomy of chromosomes 1, 7, 11, and 17 was evaluated by FISH in cervicovaginal material from 19 DES-exposed and 19 control patients.^ $17\beta$-E$\sb2, 17\alpha$-E$\sb2$, and $5\beta$-DHT-induced dramatic developmental and histological changes in the cervicovaginal tract, including hypospadia, hyperplasia, and persistent cornification. The changes induced by 17$\alpha$-E$\sb2$ were equivalent to 17$\beta$-E$\sb2.$ Neonatal 17$\alpha$-E$\sb2$-induced adenosquamous cervicovaginal tumors at 24 months. 17$\alpha$-E$\sb2$ and $5\beta$-DHT significantly increased the nuclear DNA content over control animals, but at significantly lower levels than 17$\beta$-E$\sb2.$ DNA ploidy changes were highest (80%) in animals treated neonatally and secondarily with 17$\beta$-E$\sb2.$ Secondary 17$\alpha$-E$\sb2$ and $5\beta$-DHT administration, unlike 17$\beta$-E$\sb2,$ didn't significantly increase DNA content. Chromosome 1 trisomy incidence was 66% in neonatal 17$\beta$-E$\sb2$-treated animals. Trisomy was evident in 4 DES-exposed patients: one patient with trisomy of chromosomes 1, 7, and 11; one patient with chromosome 7 trisomy; and two patients with chromosome 1 trisomy. These data demonstrated the biological effects of 17$\alpha$-E$\sb2$ and $5\beta$-DHT were age-dependent, 17$\alpha$-E$\sb2$ was equivalent to 17$\beta$-E$\sb2$ and tumorigenic when administered neonatally, and histological changes were not steroid specific. Chromosomal changes were associated with increased nuclear DNA content and chromosomal changes may be an early event in the development of tumors in human DES-exposed tissues. ^

The role of nitric oxide in apoptosis and cancer metastasis

The purpose of these studies was to investigate the role of nitric oxide (NO) in tumor metastasis. K-1735 Metastatic cells survived in blood circulation to produce experimental lung metastases, whereas nonmetastatic cells did not. After incubation with combination cytokines or lipopolysaccharide (LPS), nonmetastatic cells exhibited high levels of inducible nitric oxide synthase (iNOS) activity and NO production, whereas metastatic cells did not. The production of NO directly correlated with cytotoxic effects of cytokines or LPS. To provide direct evidence for the inverse correlation between the production of endogenous NO and the ability of K-1735 cells to survive in syngeneic mice to produce lung metastases, highly metastatic K-1735 clone 4 cells (C4.P), which express low levels of iNOS, were transfected with a functional iNOS (C4.L8), inactive-mutated iNOS (C4.S2), or neomycin-resistance (C4.Neo) genes in medium containing 3 mM NMA. C4.P, C4.Neo.3, and C4.S2.3 cells were highly metastatic whereas C4.L8.5 cells were not metastatic. The C4.L8.5 cells produced slow growing subcutaneous tumors in nude mice, whereas the other three lines produced fast growing tumors. In vitro studies indicated that the expression of iNOS in C4.L8.5 cells induced apoptosis. Collectively, these data demonstrate that the expression of recombinant iNOS in melanoma cells is associated with apoptosis, suppression of tumorigenicity, and abrogation of metastasis.^ Furthermore, multiple systemic administrations of multilamellar vesicle-liposomes (MLV) containing the lipopeptide CGP 31362 (MLV-31362) or MLV-31362 combined with murine interferon-gamma (IFN-$\gamma$) eradicated the metastases by M5076 reticular cell sarcoma. Tumor regression correlated with iNOS expression within the tumor lesions and with increased NO production. The administration of NMA significantly decreased NO production and diminished the antitumor activities. These data imply that the activation of iNOS can serve as a target for immunotherapeutic agents for treatment of murine reticulum cell sarcoma metastases. ^

Identification of the signal pathways modulated by expression of p210$\rm\sp{bcr-abl}$ and the inhibition of p210$\rm\sp{bcr-abl}$ transforming activity by polypeptides interacting with p210$\rm\sp{bcr-abl}$ in murine myeloid 32D cells

The Bcr-Abl fusion oncogene which resulted from a balanced reciprocal translocation between chromosome 9 and 22, t(9;22)(q11, q34), encodes a 210 KD elevated tyrosine specific protein kinase that is found in more than 95 percent of chronic myelogenous leukemia patients (CML). Increase of level of phosphorylation of tyrosine is observed on cell cycle regulatory proteins in cells overexpressing the Bcr-Abl oncogene, which activates multiple signaling pathways. In addition, distinct signals are required for transforming susceptible fibroblast and hematopoietic cells, and the minimal signals essential for transforming hematopoietic cells are yet to be defined. In the present study, we first established a tetracycline repressible p210$\rm\sp{bcr-abl}$ expression system in a murine myeloid cell line 32D c13, which depends on IL3 to grow in the presence of tetracycline and proliferate independent of IL3 in the absence of tetracycline. Interestingly, one of these sublines does not form tumors in athymic nude mice suggesting that these cells may not be completely transformed. These cells also exhibit a dose-dependent growth and expression of p210$\rm\sp{bcr-abl}$ at varying concentrations of tetracycline in the culture. However, p210$\rm\sp{bcr-abl}$ rescues IL3 deprivation induced apoptosis in a non-dose dependent fashion. DNA genotoxic damage induced by gamma-irradiation activates c-Abl tyrosine kinase, the cellular homologue of p210$\rm\sp{bcr-abl},$ and leads to activation of p38 MAP kinase in the cells. However, in the presence of p210$\rm\sp{bcr-abl}$ the irradiation failed to activate the p38 MAP kinase as examined by an antibody against phosphorylated p38 MAP kinase. Similarly, an altered tyrosine phosphorylation of the JAK1-STAT1 pathways was identified in cells constitutively overexpressing p210$\rm\sp{bcr-abl}.$ This may provided a molecular mechanism for altered therapeutic response of CML patients to IFN-$\alpha.$^ Bcr-Abl oncoprotein has multiple functional domains which have been identified by the work of others. The Bcr tetramerization domain, which may function to stabilize the association of the Bcr-Abl with actin filaments in p210$\rm\sp{bcr-abl}$ susceptible cells, are essential for transforming both fibroblast and hematopoietic cells. We designed a transcription unit encoding first 160 amino acids polypeptide of Bcr protein to test if this polypeptide can inhibit the transforming activity of the p210$\rm\sp{bcr-abl}$ oncoprotein in the 32D c13 cells. When this vector was transfected transiently along with the p210$\rm\sp{bcr-abl}$ expression vector, it can block the transforming activity of p210$\rm\sp{bcr-abl}.$ On the other hand, the retinoblastoma tumor suppressor protein (Rb), a naturally occurring negative regulator of the c-Abl kinase, the cellular homologue of Bcr-Abl oncoprotein, binds to and inhibits the c-Abl kinase in a cell cycle dependent manner. A polypeptide obtained from the carboxyl terminal end of the retinoblastoma tumor suppressor protein, in which the nuclear localization signal was mutated, was used to inhibit the kinase activity of the p210$\rm\sp{bcr-abl}$ in the cytoplasm. This polypeptide, called Rb MC-box, and its wild type form, Rb C-box, when overexpressed in the 32D cells are mainly localized in the cytoplasm. Cotransfection of a plasmid transcription unit coding for this polypeptide and the gene for the p210$\rm\sp{bcr-abl}$ resulted in reduced plating efficiency of p210$\rm\sp{bcr-abl}$ transfected IL3 independent 32D cells. Together, these results may lead to a molecular approach to therapy of CML and an in vitro assay system to identify new targets to which an inhibitory polypeptide transcription unit may be directed. ^