Ligand mediated activation of ectopic EGF receptor promotes matrix protein adhesion and lung colonization of rat mammary adenocarcinoma cells

Increased expression of EGF receptor (EGFR) in metastases of human mammary carcinoma as compared to cells of the primary cancer suggests a contribution of EGFR to mammary carcinoma metastasis. To test for a positive causative link, we investigated 13762NF rat mammary adenocarcinoma cloned tumor cell lines of high (MTLn3) or low (MTC) metastatic potential. While MTC cells expressed barely detectable amounts of EGFR, MTLn3 cells expressed readily detectable levels of functional receptors. A full length cDNA of the human EGFR (HER) was introduced by infection with a retroviral vector into MTC cells. Expression of HER was stable and receptors were functional with respect to surface expression, ligand binding and EGF-stimulated phosphorylation. Independent clones of the transfectants were isolated and characterized. Ligand stimulation of MTC HER cells and derived clones led to enhanced adhesion of cells to extracellular matrix proteins. Implantation of cells intravenously into female nu/nu mice revealed ligand-dependent enhancement of lung colonizing potential of EGFR-expressing cells.     

Erectile dysfunction

Peanut agglutinin (PNA) lectin-binding site patterns in primary invasive breast ductal not otherwise specified (NOS) carcinomas are related to aggressiveness of the tumor. The present study was designed to compare the expression of PNA-binding sites in the primary tumor and in local lymph node metastases. The expression of lectin-binding sites was studied using the avidin-biotin complex/immunoperoxidase technique and analyzed in relation to age of the patient and size of the breast cancer. Breast cancers and their metastases showed negativity or positivity, the latter being divided into "apical" and "non-apical" (i.e. membrane and/or cytoplasmic) depending on the main localization of staining in tumor cells. No correlation was found between primary tumors and metastases as regards PNA-binding patterns, which confirms the opinion that advanced primary tumors are polyclonal and that selected subclones of malignant cells give rise to metastases. Furthermore, the fact that primary tumors with PNA non-apical expression, a feature related to aggressiveness and poor differentiation, may have lymph node metastases with apical expression, suggests that this pattern, although no longer evident in the primary tumor, is involved in the process of cell metastasis.     

Generation of a systemic antitumor response with regional intratumoral injections of interferon gamma retroviral vector

The generation of a lasting systemic immune response is a primary goal for cancer immunotherapy. Here we examine the ability of high-titer IFN-gamma retroviral vector injected into an accessible tumor to generate significant antitumor responses at a distal untreated site. CT26 or B16F10 murine tumors were inoculated subcutaneously to form solid tumors in BALB/c or C57BL/6 mice. Seven to 10 days postinoculation, high-titer IFN-gamma retroviral vector was directly injected into the subcutaneous tumor nodule, and optimal dose and course of therapy were determined. As a model for disseminated disease, mice were inoculated intravenously with CT26 cells to form pulmonary lesions, at the same time as the subcutaneous injections. Regression of subcutaneous tumor correlated with a systemic response at the distal lung metastases in the IFN-gamma-treated group (p < 0.0005). Splenocytes from mice with completely regressed tumors had a twofold increase in percent specific cytotoxicity in a standard CTL assay as compared with nonresponding mice. CD8+ T cells were shown to be essential for the regional and systemic antitumor response, as determined by in vivo cell depletion experiments. These data demonstrate that IFN-gamma retroviral vector gene therapy delivered intralesionally can generate significant inhibition of pulmonary tumor formation distal to the treatment site. The data from these preclinical studies suggest the potential clinical value of retroviral vector-mediated cytokine gene therapy for systemic cancer.     

Recurrent T cell receptor rearrangements in the cytotoxic T lymphocyte response in vivo against the p815 murine tumor

P815 is a murine mastocytoma of DBA/2 origin which, although immunogenic, rapidly develops as a tumor in immunocompetent syngeneic hosts. In this report, we have studied, by a molecular approach, the in vivo alpha/beta T cell response to P815. Both situations of tumor growth after engraftment of naive animals or tumor rejection by preimmunized animals have been analyzed. The spectrum of T cell receptor beta chain rearrangements in the tumor-infiltrating lymphocytes was found to be highly variable among individual tumor-bearing mice. However, two rearrangements, one using V(beta)1 and J(beta)1.2 segments and one using the V(beta)1 and J(beta)2.5 segments, with conserved junctional regions, reproducibly emerge in most individuals. These two rearrangements thus correspond to "public" (recurrent) T cell clones, as opposed to "private" ones, which emerge in a seemingly stochastic fashion in immunized animals. Importantly, these public cells are observed in situations of either growth or rejection of the tumor. Quantification provides a clear increase in public T cells in secondary responses, but no obvious correlation provides between their level and primary tumor rejection. The V(beta)1- J(beta)1.2 rearrangement is borne by CTL directed against an antigen derived from P1A, a nonmutated mouse self protein which is expressed in P815 but not in normal mouse tissues except testis. A recurrent, public T cell response can thus be observed to an antigen derived from a self protein expressed by a tumor.     

HTEX4, a new human gene in the MHC class I region, undergoes alternative splicing and polyadenylation processes in testis

PURPOSE: Jasplakinolide is a novel natural product anticancer agent which acts by inducing overpolymerization of actin. The aim of the current study was to explore the activity of jasplakinolide with hyperthermia and radiation. METHODS: The response of human PC-3 and DU-145 prostate carcinoma cells and DU-145 xenografts and the response of the Lewis lung carcinoma to jasplakinolide were studied. RESULTS: Jasplakinolide was cytotoxic toward human prostate carcinoma cells, DU-145, PC-3 and LNCaP in culture, killing 1 log of cells with 0.8, 0.3 and 0.07 microM of drug in 24 h, respectively. The combination of jasplakinolide and hyperthermia resulted in primarily additive cell killing by the two modalities in the three prostate carcinoma lines. In combination with radiation, jasplakinolide produced some diminution in the shoulder of the survival curve of normally oxygenated PC-3 cells and was a radiation sensitizer of hypoxic DU-145 cells and hypoxic PC-3 cells. In vivo, jasplakinolide was an active antitumor agent against the Lewis lung carcinoma and the DU-145 prostate carcinoma xenograft. Jasplakinolide was a radiation sensitizer in the Lewis lung carcinoma. Jasplakinolide was also effective against the systemic Lewis lung carcinoma, decreasing lung metastases. Lung metastases were further decreased when jasplakinolide was administered along with radiation to the subcutaneous primary tumor. In the DU-145 tumor, the effects of jasplakinolide and fractionated radiation for 1 or 2 weeks appeared to be primarily additive. CONCLUSION: Jasplakinolide is an interesting new anticancer agent for which further study both as an anticancer agent and in combined modality regimens is warranted.     

Clonal analysis of stably transduced human epidermal stem cells in culture

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.     

Metastases from testicular carcinoma. Study of 78 autopsied cases

The necropsy records of 78 patients with histologically proved germ cell tumors of the testis, who died as a direct result of their malignant disease, were reviewed to determine the usual modes of spread, distribution of metastasis, the histologic characteristics of the metastatic foci as compared with the morphology of the primary tumor and the specific cause of death. The sites of metastases in order of decreasing frequency for all cases were lung, retroperitoneal lymph nodes, liver, mediastinal lymph nodes, brain, kidney, gastrointestinal tract, bones, adrenals, peritoneum and spleen. The absence of metastases solely in the anterior mediastinum without involvement of other mediastinal nodes (middle/posterior) strongly supports the premise for a primary extragonadal origin whenever the anterior mediastinum alone is involved with malignant disease having the histologic appearance of a primary germ cell tumor. The histologic features of the metastatic lesions were usually similar in nature to those of the primary tumor except for seminoma in which the metastatic lesions proved to be of a different histologic pattern in almost one third of the patients dying from the disease. It should be axiomatic that whenever a patient with seminoma fails to respond appropriately to radiotherapy that his treatment be immediately discontinued and that appropriate biopsies be obtained to substantiate the histologic pattern present.     

Clear cell odontogenic carcinoma with lymph node metastasis

Clear cell odontogenic tumors are rare. Review of the literature showed 9 cases with a prominent clear cell component. These lesions have exhibited an aggressive behavior characterized by an infiltrative local growth pattern, recurrence, or metastases. We report a case of an odontogenic tumor that exhibited a biphasic pattern and was characterized by lymph node involvement identical histologically to the primary tumor. We conclude that the presence of a clear cell component in an ameloblastomatous tumor should be viewed as a sign of de-differentiation, and that a malignancy with or without metastases should be considered and ruled out in such cases.     

Development of a retrovirus-based complementary DNA expression system for the cloning of tumor antigens

A new retroviral system has been developed for the generation of a cDNA library and the functional cloning of tumor antigens. These retroviral vectors contain a cytomegalovirus promoter in the 5' long terminal repeat, an extended packaging signal for rapid production of high-titer retroviral particles, and many convenient cloning sites for cDNA library construction. The vesicular stomatitis virus G protein has been used to generate pseudotype retroviral particles to enable efficient viral infection. Using this system, viral titers in the range of 10(6) colony-forming units/ml could be generated routinely, and a high transduction efficiency in human primary cells, including fibroblasts, was achieved. In addition, a new procedure has been devised for screening a retrovirus-based cDNA library without a functional selection. The utility of this system was demonstrated by constructing a retrovirus-based cDNA library and re-isolating the NY-ESO-1 tumor antigen from a cDNA library using an antigen-specific CTL. This approach can facilitate the identification of novel tumor antigens recognized by T cells without knowledge of MHC class I restriction elements and is generally applicable for the isolation of any gene as long as a biological assay is available.     

p53 nuclear protein overexpression in colorectal cancer: a dominant predictor of survival in patients with advanced hepatic metastases

PURPOSE: To determine whether p53 protein expression is similar within primary colorectal cancer (CRC) and synchronous regional and distant metastases and to assess whether p53 nuclear protein expression could predict outcome in patients with synchronous unresectable liver metastases treated by hepatic artery infusional (HAI) chemotherapy. MATERIALS AND METHODS: Paraffin sections from tumor and corresponding normal mucosa representative of 50 consecutive advanced CRC cases were examined for p53 nuclear protein expression by immunohistochemistry using the monoclonal antibody PAb 1801. Patterns of p53 nuclear expression were correlated with standard clinicopathologic variables and outcome, including response to HAI and survival. In a subset analysis, the pattern of nuclear p53 immunoreactivity was compared between primary CRC and lymph node and liver metastases. RESULTS: Positive nuclear immunoreactivity for p53 protein was found in 72% of cases. The pattern of p53 protein expression in lymph node and liver metastases was identical to that of the primary tumor. The median survival time was 21.0 months in patients with p53-positive tumors and 53.2 months in patients with p53-negative tumors (Wilcoxon test P = .038). Two-year survival rates were 41.7% and 78.6%, respectively (P < .01). No significant difference was found in the response rates to HAI chemotherapy between the two groups. By multivariate analysis, p53 protein status was the single best predictor of survival, with a relative risk of 6.312. CONCLUSION: Our results indicate that nuclear p53 protein status in primary CRC is similar to that in metastatic sites and may be the dominant predictor of survival in patients with advanced hepatic metastases.     

In vitro growth and drug sensitivity of tumor colony-forming units from human tumor xenografts

To investigate the feasibility of using tissue obtained from human tumor xenografts for in vitro screening of antineoplastic agents, we grew human tumor colony-forming units (CFU) in semisold agar from xenografts serially passaged in nude mice. Growth of human tumor CFU was accomplished from nine xenografts representing five different histological tumor types (ovarian carcinoma, adenocarcinoma of the colon, malignant melanoma, epidermoid carcinoma of the lung, and malignant astrocytoma). Cloning efficiency ranged from 0.04 to 0.1% and showed significant variability both between tumor types and between individual animals bearing the same type of xenograft. A high percentage of tumor CFU was in S phase [47 +/- 20% (S.D.)] as determined by the thymidine "suicide" technique. The number of tumor CFU observed increased linearly with increasing numbers of cells plated. In vitro drug sensitivity of the tumor CFU was assessed to Adriamycin, cis-platinum, and melphalan. The patterns of drug sensitivity were found to be reproducible and stable over a period of 9 months. Drug sensitivity curves to Adriamycin for five xenografts representing four tumor types showed complex patterns with plateau portions similar to those described for tumor CFU from primary tumors. The rank order of sensitivity of the tumors was compared to that of normal granulocyte-macrophage progenitors and, with the exception of the melanomas, was found to correlate well with clinical experience (order of sensitivity = colon less than ovary less than bone marrow). Growth of human tumor CFU from xenografts represents a reproducible and stable means for the study of the biology of tumor CFU and has potential applications as a means for screening new anticancer agents.     

Administration of recombinant interleukin 12 prevents outgrowth of tumor cells metastasizing spontaneously to lung and lymph nodes

The present study investigates the ability of recombinant interleukin 12 (rIL-12) to modulate the growth of a primary tumor as well as the outgrowth of metastatic tumor cells in an ovarian carcinoma (OV-HM) model. This aggressive tumor displayed rapid growth of the primary tumor mass, high incidence of metastases to lung and lymph nodes, and invasion from the primary s.c. site to the peritoneal cavity. Starting 12 days after s.c. tumor cell implantation, several i.p. injections of rIL-12 at 2-3 day intervals resulted in regression of growing tumors. These treated mice did not show signs of metastases or tumor recurrence at the original site. One month after tumor implantation, untreated mice did not have visible lung metastasis, but some did have palpable lymph nodes. At this stage, the primary tumors of animals without palpable lymph nodes were surgically resected. When examined 2 months later, most animals had developed lymph node and lung metastases. In contrast, rIL-12 injections after tumor resection inhibited the development of metastases in both lung and lymph nodes. This contrasted with the failure of IL-2 to prevent metastases. Even for mice already showing signs of lymph node metastases or invasion of the abdominal wall, rIL-12 administration after tumor resection prevented further invasion to the peritoneal cavity and growth of metastatic tumor cells in lung. It was somewhat surprising that the IL-12 treatment of animals after 1 month of tumor growth without resection also resulted in complete tumor regression, as well as eradication of micrometastasis that would have occurred before the treatment. Moreover, they exhibited resistance to a rechallenge with the same tumor but not with a second tumor. Thus, this tumor system provides a relevant model to clinical situations in terms of treatment of advanced tumors and metastases. These results also indicate that IL-12 can induce a curative immune response, even in the face of an aggressive micrometastasizing tumor.     

Expression of glycylated tubulin during the differentiation of spermatozoa in mammals

Local invasion and metastatic spread to distant sites are major causes of death in patients with malignant pheochromocytoma. Since appropriate in vivo models do not exist, little is known about the underlying mechanisms of tumor growth and invasion. We, therefore, developed an animal model of malignant pheochromocytoma and established organotropic metastatic variants of PC12 rat pheochromocytoma cells. PC12 cells were established as xenografts to BALB/c NCR-NU mice. Subsequent to development of tumors or metastases, primary cultures from local tumors, metastases to lymph nodes, lungs and liver were established. These were subcultured in vitro and reinjected for up to five successive in vivo/in vitro cycles. Xenografted PC12 cells grew tumors with a doubling time of 6.78 +/- 0.58 days during log phase of tumor growth, killing hosts within 5-12 weeks depending on the experimental conditions. Tumors reproducibly metastasized to lymph nodes and the lung. Spontaneous metastases to the liver were not observed, but were achieved by intrasplenic injection of parent PC12 cells. In vitro, the metastatic cell lines displayed striking differences in morphology, overall growth patterns and nutritional requirements as well as binding to purified extracellular matrix proteins compared to the parent cell line. In vivo, the metastatic variants showed marked enhancement of metastatic ability. This is the first report of PC12 rat pheochromocytoma cells to exhibit the malignant phenotype in vivo. We also established variant PC12 cell lines that preferentially metastasized to specific sites and that had acquired different in vitro behavior and ability to metastasize. This unique model system should be useful for further studies relating to the invasion and metastases of pheochromocytoma and may prove valuable for investigations of novel antineoplastic therapies in vitro and in vivo.     

Gene therapy for primary and metastatic pancreatic cancer with intraperitoneal retroviral vector bearing the wild-type p53 gene

BACKGROUND: Metastatic pancreatic cancer is uniformly fatal because no effective chemotherapy is available. Mutations in the p53 tumor suppressor gene are found in up to 70% of pancreatic adenocarcinomas. We examined the efficacy of a retroviral vector containing the wild-type p53 gene on metastatic pancreatic cancer in a nude mouse model. METHODS: Bxpc3 human pancreatic cancer cells were transduced with either a retroviral p53 vector or an LXSN empty vector. Cells were examined for incorporation of tritiated thymidine to determine the effect of p53 retroviral transduction on DNA synthesis, and a TACS2 assay for apoptosis was performed. The functional activity of p53 in transduced cells was assessed by Western blot analysis with an antibody to WAF1/p21. In vivo effects of intraperitoneal injections of the p53 vector were examined in a nude mouse model of peritoneal carcinomatosis. RESULTS: Cells treated with the p53 vector exhibited a 59% to 85.5% reduction in cell number compared with the control cells (P < .05). p53-treated cells demonstrated decreased incorporation of tritiated thymidine (12.7% +/- 0.7% vs 17.5% +/- 1.4%; P = .002), increased staining for apoptosis, and increased expression of the WAF1/p21 protein. Treatment of nude mice with the retroviral p53 vector resulted in a significant inhibition of growth of the primary pancreatic tumor, as well as the peritoneal tumor deposits, compared with the LXSN control vector. CONCLUSIONS: Intraperitoneal delivery of a retroviral p53 vector may provide a novel treatment approach for peritoneal carcinomatosis from pancreatic cancer.     

Retrovirus-mediated transfer of the human alpha-L-iduronidase cDNA into human hematopoietic progenitor cells leads to correction in trans of Hurler fibroblasts

Hurler syndrome (mucopolysaccharidosis IH or MPS IH) is a congenital mucopolysaccharide storage disorder resulting from a genetic deficiency of alpha-L-iduronidase (IDUA), which is required for lysosomal degradation of glycosaminoglycans heparan sulfate and dermatan sulfate. Even though histocompatible bone marrow transplantation has been applied for the treatment of Hurler syndrome, gene therapy via autologous bone marrow transplantation (BMT) may be more beneficial for this disease. Two retroviral vectors containing a full-length human IDUA cDNA were constructed using Moloney murine leukemia virus (MoMLV)-based vector backbones. High-titer vector-producing clones containing the L-HuID-SN and MFG-HuID retroviral vectors were established. The efficiency of gene transfer into primitive human CD34+ hematopoietic cells using both retroviral vectors is in the range of 18-23%. The level of enzyme expression in transduced primary bone marrow cells was increased 40- to 50-fold compared with that of sham-transduced cells. Enzyme produced by the progeny of the transduced human CD34+ cells carrying IDUA cDNA corrected Hurler fibroblasts via mannose-6-phosphate receptors. These findings suggest that genetically modified hematopoietic progenitor cells can potentially be useful for gene therapy of Hurler syndrome.     

Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotides on human melanoma cells in vitro and and in mice

BACKGROUND: Phosphorothioate oligodeoxynucleotides ([S]ODNs) contain a modified internucleoside phosphate backbone. Antisense [S]ODNs targeted to specific oncogenes have been used with some therapeutic success in animal models human leukemia; however, the potential for antisense [S]ODN treatment of solid tumors has only recently been explored. Purpose: We evaluated the effects of antisense [S]ODNs targeted to the c-myc oncogene on the proliferation of human melanoma cells in vitro and on the growth of human melanoma xenografts in CD-1 nude (nu/nu) mice, METHODS: The effects of 15-mer [S]ODNs containing c-myc sense, c-myc antisense, and two different scrambled sequences on the proliferation and viability of cultures of three established human melanoma cell lines (M14, JR8, and PLF2) were determined by measuring cell numbers and use of the trypan blue exclusion test. The induction of apoptosis in these cells following treatment with [S]ODNs was evaluated by fluorescence-activated cell sorter (FACS) analysis. FACS analysis was also used to determine the effects of [S]ODN treatment on the proliferation of primary cultures of a human melanoma explant (NG cells). The expression of c-Myc protein in cultured NG cells after treatment with [S]ODNs was examined by western blot analysis. The antitumor activity and the toxic effects of several [S]ODN treatment regimens were monitored by measuring differences in tumor weight (percent tumor weight inhibition), tumor growth rate (tumor growth inhibition), animal lifespan (percent increase in lifespan), the number of toxic deaths and the median number of long metastases in treated and control mice bearing NG xenografts. c-Myc protein expression in NG tumor cells following [S]ODN treatment was evaluated by FACS analysis, and the extent of apoptosis in these cells was determined by FACS analysis and morphologic examination. RESULTS: Treatment with antisense [S]ODNs, but not the others, inhibited the growth of all tested melanoma cultures in vitro; FACS analysis revealed that growth inhibition was associated with the induction of apoptosis. Antisense [S]ODN treatment also led to reduced celluLar levels of c-Myc protein. In vivo, [S]ODN antitumor activity and toxicity were dose and schedule dependent; however, only antisense [S]ODNs exhibited antitumor activity. Mice bearing NG xenografts treated with antisense [S]ODNs showed a marked inhibition of tumor growth, a reduction in the number of long metastases, and an increase in life span. Reduced levels of c-Myc protein and increased levels of apoptosis were also observed in NG tumor cells following antisense [S]ODN treatment. CONCLUSIONS: treatment of human melanoma cells and solid tumors with antisense [S]ODNs targeted to c-Myc inhibits their growth and is associated with the induction of apoptosis.