Isolation and characterization of C-type viral gene products of virus-negative mouse cells.

Antigens which immunologically cross-react with two mouse C-type viral polypeptides, p30 and p12, are present at very low levels in normal virus-negative mouse cells. These two antigens have been purified by 50-300-fold from cell extracts and shown to cochromatograph with the corresponding labeled viral polypeptides in several systems. Their type-specific antigenicities are shown to be distinct from those of previously tested MuLV isolates suggesting that they may be components of a new class of endogenous C-type virus. The methods utilized in the present studies for concentration of virus-specific antigens of normal mouse cells provide an approach for detection of C-type viral antigens in cells of other species.

The tat gene of human T-lymphotropic virus type 1 induces mesenchymal tumors in transgenic mice.

Human T-lymphotropic virus type 1 (HTLV-1) is a suspected causative agent of adult T-cell leukemia. One of the viral genes encodes a protein (tat) that not only results in transactivation of viral gene expression but may also regulate the expression of certain cellular genes that are important for cell growth. Transgenic mice that expressed the authentic tat protein under the control of the HTLV-1 long terminal repeat were generated, and cell types that are permissive for the viral promoter and the effects of the tat gene on these cells were studied. Three of eight founder mice with high levels of expression of the transgene in muscle were bred and then analyzed. All developed soft tissue tumors at multiple sites between 13 to 17 weeks of age. This phenotype was transmitted to nine of nine offspring that inherited the tat gene and were available for analysis. The remaining five founders expressed the transgene in the thymus, as well as in muscle. This second group of mice all exhibited extensive thymic depletion and growth retardation; in all of these mice, death occurred between 3 to 6 weeks of age before tumors became macroscopically visible. The tat gene under the control of the HTLV-1 regulatory region showed tissue-specific expression and the tat protein efficiently induced mesenchymal tumors. The data establish tat as an oncogenic protein and HTLV-1 as a transforming virus.

A transgenic mouse model for human neurofibromatosis.

Human T-lymphotropic virus type 1 (HTLV-1) has been associated with the neurologic disorder tropical spastic paraparesis and possibly with multiple sclerosis. The tat gene of HTLV-1 under control of its own long terminal repeat is capable of inducing tumors in transgenic mice. The morphologic and biologic properties of these tumors indicate their close resemblance to human neurofibromatosis (von Recklinghausen's disease), the most common single gene disorder to affect the nervous system. The high spontaneous incidence of this disease, together with the diverse clinical and pathologic features associated with it, suggests that environmental factors may account for some of the observed cases. Multiple tumors developed simultaneously in the transgenic tat mice at approximately 3 months of age, and the phenotype was successfully passed through three generations. The tumors arise from the nerve sheaths of peripheral nerves and are composed of perineural cells and fibroblasts. Tumor cells from these mice adapt easily to propagation in culture and continue to express the tat protein in significant amounts. When transplanted into nude mice, these cultured cells efficiently induce tumors. Evidence of HTLV-1 infection in patients with neural and other soft tissue tumors is needed in order to establish a link between infection by this human retrovirus and von Recklinghausen's disease and other nonlymphoid tumors.

Sentinel lymph node mapping in gynecologic malignancies.

Lymph node status is the most important prognostic factor for women with vulvar, cervical and endometrial carcinoma and complete lymph node dissection has historically been an integral part of the surgical treatment of these diseases. Lymphadenectomy can be morbid for patients, who may experience wound breakdown, lymphocyst formation or chronic lymphedema, among other problems. Sentinel lymph node mapping is a newer technology that allows selective removal of the first node draining a tumor thereby allowing a potentially less aggressive procedure to be performed. Sentinel node mapping is well accepted for the management of breast carcinoma and cutaneous melanoma, and has resulted in reduced morbidity without adversely affecting survival. Sentinel node mapping is currently being investigated for treatment of gynecologic cancers. Recent studies show promise for incorporating the sentinel node mapping technique for treatment of several gynecologic malignancies.

Insulin-like growth factor I receptors in normal and neoplastic human endometrium.

Insulin-like growth factor I (IGF-I) binding sites were characterized in normal and neoplastic endometrium. The characteristics of the endometrial IGF-I receptor are similar to those reported for other tissues. The binding of 125I-IGF-I to the endometrial membranes is saturable and time, temperature, and pH dependent. The 125I-IGF-I binding activity to the membranes obtained from differentiated and undifferentiated adenocarcinoma as well as sarcoma of the endometrium was significantly higher (P less than 0.05) when compared to the binding activity of the membranes obtained from normal endometrium. The Scatchard analysis of the competitive binding data of both normal and neoplastic endometrium revealed linear plots. This indicated a single class binding site for IGF-I with equilibrium dissociation constants (Kd) of 5.0, 6.8, 6.94, and 6.88 nM for normal, differentiated, and undifferentiated adenocarcinoma, and sarcoma of the endometrium, respectively. Therefore, the differences observed in 125I-IGF-I binding between normal and neoplastic endometrial membranes was due to an increase in the number of IGF-I binding sites and not to a change in receptor binding affinity. Autoradiograms from affinity labelling studies revealed a band corresponding to Mr 132,000 subunit of the receptor which is characteristic of the type I receptor reported for other tissues. A dimer of the alpha subunit (Mr 263,000) was also observed in all four categories of endometrial tissue. Additionally, autoradiograms obtained from sarcoma of the endometrium revealed a Mr 40,000 band that was only displaced by IGF-I and IGF-II peptides but not by the monoclonal antibody alpha IR-3 to the type I receptor. These suggest that the band is representative of the IGF-I or IGF-II binding protein. A similar band was not observed in the other tissues. The results show that the human endometrium contains high affinity IGF-I or IGF-II binding sites. The fact that IGF-I binding activity was significantly higher for neoplastic endometrium suggests that IGF-I may play an important role on supporting the growth of this neoplastic tissue.

A modified p53 overcomes mdm2-mediated oncogenic transformation: a potential cancer therapeutic agent.

The antiproliferative activities of wild-type (wt) p53 are inhibited by mdm2 (murine double minute2) oncogene product. We tested growth suppression activity of p53 14/19, an engineered p53 variant, which does not bind mdm2 and is completely resistant to the inhibition by mdm2. p53 14/19, unlike wt p53, suppressed the growth of cancer cells that contain amplified mdm2 oncogene efficiently by direct DNA transfection or adenovirus-mediated gene transfer. In addition, p53 14/19 also inhibited the growth of several different cancer cell lines expressing low levels of mdm2 oncogene product as efficiently as wt p53. We further examined the antioncogenic potencies of p53 14/19 in the rat embryo fibroblast cotransformation assay. Addition of wt p53 failed to cause any significant decrease in ras plus mdm2 foci counts. In contrast, cotransfection of p53 14/19 with ras and mdm2 significantly reduced foci number. In similar experiments, cotransfection of wt p53 or 14/19 p53 resulted in significant inhibition of oncogenic transformation in rat embryo fibroblast mediated by an activated ras plus c-myc, adenovirus E1A, or human papillomavirus E7 oncogenes. Therefore, these results suggest that p53 14/19 modified tumor suppressor gene may be a promising therapeutic agent for human cancers that express abnormally high levels of mdm2 oncogene product.

Inhibition of constitutively active Stat3 suppresses growth of human ovarian and breast cancer cells.

Signal transducers and activators of transcription (STATs) are transcription factors activated in response to cytokines and growth factors. Constitutively active Stat3 has been shown to mediate oncogenic transformation in cultured cells and induce tumor formation in mice. An increasing number of tumor-derived cell lines as well as samples from human cancer have been reported to express constitutively active Stat3 protein. We previously demonstrated that ovarian cancer cell lines express high levels of constitutively active Stat3. In this study, we show that inhibition of the Stat3 signaling pathway using the Janus Kinase-selective inhibitor, AG490, and a dominant negative Stat3 (Stat3beta) significantly suppresses the growth of ovarian and breast cancer cell lines harboring constitutively active Stat3. In the ovarian cancer cell lines, AG490 also diminished the phosphorylation of Stat3, Stat3 DNA binding activity, and the expression of Bcl-x(L). Further, AG490 induced significant apoptosis in ovarian and breast cancer cell lines expressing high levels of constitutively active Stat3 but had a less profound effect on normal cells lacking constitutively active Stat3. AG490 also enhanced apoptosis induced by cisplatin in ovarian cancer cells. These results suggest that inhibition of Stat3 signaling may provide a potential therapeutic approach for treating ovarian and breast cancers.

Prevention of cervix cancer.

Cervix carcinoma is an important health problem world-wide, being the second most common cancer among women, ranking first in many developing countries. A number of important epidemiological risk factors have been identified as contributing to the development of CIN and invasive cervix carcinoma. Of key importance is infection with human papillomavirus (HPV), which is the primary risk factor. There are evolving primary and secondary preventive strategies that could further reduce the burden from cervical carcinoma. The possible primary preventive strategies include risk reduction, diet or dietary supplements, HPV vaccines, and other chemopreventive agents. The possible advances in secondary preventive strategies include new technologies for Pap smears, HPV typing triage, and other adjuvant screening procedures. The impact of these strategies will depend upon evidence to support their use along with the characteristics of the population and environment in which they are used.

Elevated phosphorylation of AKT and Stat3 in prostate, breast, and cervical cancer cells.

We examined whether the persistent activation of AKT or Stat3 oncogene product is present in prostate, breast, and cervical cancer cells. We found that some prostate and breast cancer cell lines express high levels of phosphorylated AKT. Interestingly, the cancer cells, which only express low levels of phosphorylated AKT express high levels of phosphorylated Stat3. AKT or Stat3 is also highly phosphorylated in human papilloma virus-negative cervical cancer cells. Therefore, these results indicate that AKT and Stat3 are highly phosphorylated in some breast, prostate and cervical cancer cells, which may play a role in tumorigenesis of these cancers.

Growth suppression activity of the PTEN tumor suppressor gene in human endometrial cancer cells.

The AKT proteins are constitutively activated in several types of human cancers, which may play a role in carcinogenesis. In this study, we examined the activation of AKT in a panel of human endometrial cancer cell lines and tumor samples in this study. Two endometrial cancer cell lines, Ishikawa (ISK) and RL-95 and several tumor samples showed elevated levels of phosphorylated AKT PTEN, which is mutated in 45% of endometrial cancers, is a negative regulator of AKT. We examined the growth suppression activity of PTEN in ISK and KLE endometrial cancer cells. Expression of PTEN significantly suppressed the growth of both cell clines. In primary rat embryo fibroblasts, PTEN also inhibited malignant transformation mediated by ras and c-myc oncogenes. These two oncogenes are commonly mutated or amplified in endometrial cancer. These results suggest that PTEN may be a potent therapeutic agent for endometrial cancer.

A matched study of surgically treated stage IB adenosquamous carcinoma and adenocarcinoma of the uterine cervix.

Thirty-eight patients with surgically treated stage IB adenosquamous carcinoma of the uterine cervix (AS) have been matched with patients with other histologic subtypes of adenocarcinoma (A) for stage, lesion size, node status, grade of adenocarcinoma and age at diagnosis. An additional six patients with AS were unable to be matched. Overall 5-year survival and disease-free survival for the matched AS and A were not significantly different, 83 vs. 90%, and 78 vs. 81% nor were the number of recurrences, 8/38 AS vs. 6/38 A, but the mean time to recurrence was significantly shorter in the AS group: 11 vs. 32 months (P = 0.003). A subgroup of AS with a high risk of a poor outcome can be identified based on either lesion size >/= 4 cm, depth of invasion >/= 10 mm or plevic lymph node metastasis. These patients may be suitable candidates for adjuvant therapy before or after surgical treatment.

Treatment of refractory ovarian carcinoma with paclitaxel and cisplatin after treatment failure with single-agent paclitaxel.

The clinical response to paclitaxel and cisplatin was evaluated in fifteen patients with refractory epithelial ovarian cancer who failed to respond to treatment with single agent paclitaxel. Patients received combination chemotherapy every 3 weeks with both 135 mg/m2 (9) or 175 mg/m2 (6) of paclitaxel and 50 mg/m2 (2), 75 mg/m2 (4) or 100 mg/m2 (9) of cisplatin. There was 1 complete clinical response, with 2 partial clinical responses for an overall response rate of 20%. The progression free interval was 6+ months for the complete responder and 9.5+ months for the partial responders. Overall five (33%) patients experienced an improvement in clinical response over that seen with paclitaxel alone, and 5 patients have died. Improvement in clinical response with combination chemotherapy compared to paclitaxel alone was positively associated with the cisplatin dose; while disease progression and death were inversely associated with the paclitaxel dose. Addition of cisplatin to paclitaxel may be useful in the treatment of patients who fail to respond to paclitaxel alone.

Potential use of surface-active agents for controlling Mycoplasma contamination in animal cell cultures.

Seven nonionic detergents, which were determined to be relatively nontoxic to selected animal cell cultures, were tested for their lethal effect on the GDL strain of Mycoplasma hyorhinis. Of the seven detergents tested, five were found to cause complete lysis of the organism in vitro within 24 hr at 37 C. These detergents included Triton WR-1339 and Tweens 20, 40, 60, and 80. When different concentrations of the detergents were tested, Tween 80 was found to be the most effective and Triton WR-1339 the least effective in lysing the mycoplasmata. These same five detergents were used to treat a rat nephroma cell line which was chronically infected with the GDL strain. The mycoplasmata were eliminated from those cultures treated with Triton but they persisted in cultures exposed to the Tween compounds. The Triton-treated cells remained free from infection over a 7-month period, as determined both by cultural methods and fluorescent-antibody staining. The "cure" was effected by treating the cells for either 48 hr with maintenance media containing 1 mg of Triton per ml or for 96 hr with a concentration of 500 mug/ml. Triton was also effective in eliminating the GDL, strain from experimentally infected rat embryo cells after a 48-hr treatment with a concentration of 1 mg/ml. Four other species of Mycoplasma, which were completely lysed by Triton in vitro, were not eliminated from experimentally infected cells by a single treatment with Triton, although the severity of the infection was apparently reduced.

Characterization of morphologic revertants of murine and avian sarcoma virus-transformed cells.

Morphologic revertants which contain avian or murine sarcoma viruses have previously been isolated at low frequency from clonal lines of transformed mammalian cells. In the present study, these lines have been further characterized. They are indistinguishable from nontransformed parent cell lines with respect to parameters such as saturation density and colony formation in depleted medium or on monolayers of contact-inhibited cells. The rate of glucose uptake had also reverted to normal. The malignant potential of one of the revertant lines was examined and found to be markedly reduced compared to that of the corresponding transformed cells. The differences in the susceptibilities of revertant cells to retransformation by the same or other oncogenic viruses suggest that different cellular genes may be involved in expression of transformation by various tumor viruses.

Constitutive activation of stat 3 oncogene product in human ovarian carcinoma cells.

OBJECTIVE: Stat 3 functions in transducing signals from the cell's surface to its nucleus and activation of gene transcription. Aberrations of Stat 3 in breast cancer have raised the possibility of its contribution to oncogenesis. Our goal was to examine ovarian cancer cell lines to determine whether Stat 3 plays a relevant role in ovarian carcinogenesis. METHODS: Protein lysates were extracted from normal ovarian surface epithelial cells and malignant cells. Western blotting techniques were performed with phosphorylation-independent or phosphorylation-specific Stat 3 (tyrosine 705) antibody. Confirmation of Stat 3 activation was determined by a luciferase reporter driven by a promoter containing Stat 3-specific binding sites. Bcl-x(L) and cyclin D(1) were also analyzed by Western blotting. RESULTS: MDAH 2774, OV-1063, Caov-3, and O.C. 22819 expressed high levels of phosphorylated Stat 3. In contrast, A2780 and normal ovarian surface epithelial cells had little Stat 3 phosphorylation recognized. Confirmation of persistent activation of Stat 3 activity was shown by transfection of cells with a Stat 3 luciferase reporter. Potential downstream mediators of Stat 3 including Bcl-x(L) and cyclin D(1) were also evaluated. In cells expressing activated Stat 3, high levels of both Bcl-x(L) and cyclin D(1) were detected, whereas in A2780 cells, which did not express activated Stat 3, only low levels of Bcl-x(L) and cyclin D(1) were expressed. CONCLUSIONS: Constitutive activation of Stat 3 is present in ovarian cancer lines but not in normal ovarian surface epithelial cells. Activation of Stat 3 is a common event during oncogenic transformation upstream to both Bcl-x(L) and cyclin D(1). The relationship of this aberrancy of ovarian carcinoma harboring activated Stat 3 deserves further investigation.

Cultured endometrial cancer cells exhibit autocrine growth factor stimulation that is not observed in cultured normal endometrial cells.

OBJECTIVE: To evaluate the autocrine stimulation hypothesis, primary cultures of malignant and normal endometrium were assayed for differences in response to growth factors (GF) GF and receptor blocking antibodies. METHODS: Thirteen normal and 10 malignant endometrial samples were collected. Cells were enzymatically dispersed and maintained in serum-free medium. They were incubated with epidermal GF (EGF), transforming GF-alpha (TGF-alpha), insulin-like GF-I (IGF-1), anti-EGF receptor antibody (Ab528), and anti-IGF-1 receptor antibody (alpha IR3) at physiologic concentrations. Tritiated thymidine incorporation was measured. RESULTS: Malignant endometrial cells increased thymidine incorporation when incubated with EGF (20.75%), TGF-alpha (19.8%), or IGF-1 (32.8%) compared to untreated control cells. When incubated with Ab528 or alpha IR3 antibodies alone, proliferation of malignant cells was inhibited (-12.4 and -23%, respectively, P < 0.003). Normal endometrial cells were inhibited by EGF (-24.9%), TGF-alpha (-25.6%), and IGF-1 (31.9%). Incubation of normal cells with Ab528 and alpha IR3 antibodies stimulated growth (125 and 115%, respectively, P < 0.02). CONCLUSIONS: These data are consistent with the autocrine stimulation hypothesis for neoplastic endometrium and illustrate differences compared to nonneoplastic endometrial growth factor-mediated proliferation.

Transforming growth factor-alpha and insulin-like growth factor-I, but not epidermal growth factor, elicit autocrine stimulation of mitogenesis in endometrial cancer cell lines.

OBJECTIVES: Endometrial carcinoma cell lines were evaluated for epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha), and insulin-like growth factor I (IGF-1) production and for autocrine stimulation. METHODS: Conditioned, serum-free media (CM) from cell lines RL95-2, KLE, HEC, and Ishikawa (ISH) were concentrated radioimmunoassayed (RIA). Samples for the IGF-1 assay were extracted with acid-ethanol to remove IGF-1 binding protein. Polymerase chain reaction (PCR) was used to validate the presence of mRNA for growth factors and receptors. Cells were incubated with Ab528, an antibody blocking EGF receptors, and alphaIR3, an antibody blocking IGF-1 receptors. Proliferation was quantified using [3H]thymidine incorporation. RESULTS: TGF-alpha was detected in CM: RL95-2 (0.4 +/- 0.001 ng/ml), KLE (0.7 +/- 0.003 ng/ml), HEC (0.8 +/- 0.01 ng/ml), ISH (1.2 +/- 0.05 ng/ml). No EGF was detected in CM. In extracted samples, IGF-1 was detected in CM: RL95-2 (0.8 +/- 0. 03 ng/ml), KLE (1.25 +/- 0.02 ng/ml), HEC (1.6 +/- 0.01 ng/ml), ISH (1.6 +/- 0.08 ng/ml). Unconditioned media served as the control. EGF, TGF-alpha, and IGF-1 mRNA was identified in all cell lines, as was the mRNA for EGF and IGF-1 receptors. Incubation with Ab528 or alphaIR3 resulted in significant inhibition of DNA synthesis in HEC 1A, KLE, and ISH. No inhibition was detected in the RL95-2 cell line. A control antibody did not inhibit the cell lines. CONCLUSION: Autocrine production and stimulation of endometrial carcinoma cell lines by TGF-alpha and IGF-1 are demonstrated in three of four endometrial cancer cell lines. No measurable EGF was produced by any of the cell lines.

Comparisons of the immunological properties of two structural polypeptides of type C RNA viruses endogenous to old world monkeys.

Immunologically very closely related type C RNA viruses are endogenous to the domestic cat and to an old world primate, the baboon. In the present studies, radioimmunological techniques have been developed for detection of the 15,000 and 30,000 molecular weight (MW) polypeptides of each virus. The much more pronounced type-specific antigenic determinants of the lower MW polypeptides made it possible to readily differentiate these viruses from each other as well as from a type C virus isolate from a second baboon species. Normal rhesus monkey tissues were partially purified and shown to contain a reactivity with MW and immunological properties similar to that of the baboon virus 30,000 MW polypeptide. Despite a similar degree of purification, antigenic reactivity like that of the baboon virus 15,000 MW polypeptide was undetectable even in the brodest immunological tests available for this polypeptide. The present findings indicate that the immunological properties of two structural polypeptides of closely related viruses endogenous to primate and feline species have undergone different rates of antigenic change in the course of evolution within their respective host cell genome.

Combined carboplatin and cisplatin. Limited prospects for dose intensification.

BACKGROUND: The relative lack of overlapping toxicities and less-than-complete cross-resistance of tumors treated with both carboplatin and cisplatin may allow these two analogues to be given in combination to exploit platinum dose intensity therapeutics. Early experience with combined platinum regimens, however, found myelosuppression, particularly severe thrombocytopenia, to be dose-limiting. It was postulated that a 2-day interval between carboplatin and cisplatin would allow for near complete clearance of the former before cisplatin administration and a potential gain in dose intensity. METHODS: Other carboplatin-cisplatin regimens produced Grade 3-4 toxicity in 20% of patients. By defining 95% confidence limits around this observed rate of Grade 3-4 toxicity, the accrual needs of this study were determined in a two-stage process. Sixteen patients with advanced malignancies were entered onto a trial of 300 mg/m2 of carboplatin on day 1, followed by 125 mg/m2 of cisplatin on day 3 every 28 days. Hematologic and nonhematologic toxicity was closely monitored, including the use of serial audiograms, to allow appropriate dose modification. RESULTS: A total of 40 courses of combination platinum therapy was administered to 15 patients who were evaluable for toxicity. Higher-than-anticipated ototoxicity and neurosensory toxicity was observed. WHO Grade 3 ototoxicity (hearing loss) was documented in 12 of 15 patients (80.0%, 95% confidence interval [CI]: 52.0-97.0%) and emerged as the dose-limiting side effect of this regimen. High-frequency hearing loss, as demonstrated by conventional audiograms, was universal among all 12 patients who received at least 2 courses of combination platinum therapy (100%, 95% CI: 73.5-100%). Grade 2 or 3 neurosensory toxicity also was observed in 4 of 15 patients. Hematologic toxicity was manageable. WHO Grade 3-4 neutropenia or thrombocytopenia occurred in only 14% and 11%, respectively, of 40 courses. There was no evidence of cumulative marrow toxicity. Calculated dose intensities (mg/m2/week) were 94 +/- 26.0 for carboplatin, 39.3 +/- 12.4 for cisplatin, and 64.0 +/- 19.2 for the combination (expressed as cisplatin equivalents). Objective responses (complete response+partial response) occurred in 8 of 16 subjects (50.0%, 95% CI: 24.7-75.4%), with 1 patient achieving a complete response of 14+months. CONCLUSIONS: The schedule of day 1 carboplatin plus day 3 cisplatin every 4 weeks appeared to allow a higher platinum dose intensity with less myelotoxicity than previously reported schedules combining these two analogues. Ototoxicity, however, was unexpectedly severe and limits future prospects for the use of combined platinum analogues to achieve dose intensification.