Enhanced apoptosis with combination C225/radiation treatment serves as the impetus for clinical investigation in head and neck cancers.

PURPOSE: Epidermal growth factor receptor (EGFr) is overexpressed in a majority of head and neck squamous cell carcinomas, and this overexpression is associated with a poor prognosis. Therefore, EGFr has become the target of investigations aimed at disabling the receptor to determine whether this process leads to improved tumor kill with conventional treatment. MATERIALS AND METHODS: C225 is an anti-EGFr monoclonal antibody that inhibits receptor activity by blocking the ligand binding site. A panel of human head and neck squamous cell carcinoma cell lines was used to study the combination of C225 and radiation. RESULTS: It was determined that the combination of C225 (5 microgram/mL) delivered simultaneously with radiation (3 Gy) resulted in a greater decrement in cellular proliferation than either treatment alone. This reduction in proliferation correlated with reduced EGFr tyrosine phosphorylation and a reduction in phosphorylated signal transducer and activator of transcription-3 (STAT-3) protein (known to protect cells from apoptosis). Also, the decrement in proliferation correlated with increased apoptotic events, thereby indirectly linking C225/radiation-induced regulation of STAT-3 protein to apoptosis. CONCLUSION: This preclinical work serves as important support for the ongoing clinical investigation of C225 and radiotherapy for patients with head and neck carcinomas. The initial results of these clinical studies have been promising.

Paclitaxel derivatives for targeted therapy of cancer: toward the development of smart taxanes.

The pharmacologic efficacy of the promising antitumor agent paclitaxel (Taxol) may be potentially enhanced through derivatization of the drug to a water-soluble tumor-recognizing conjugate. This work reports the design and synthesis of the first tumor-directed derivative of paclitaxel. A 7-amino acid synthetic peptide, BBN[7-13], which binds to the cell surface bombesin/gastrin-releasing peptide (BBN/GRP) receptor, was conjugated to the paclitaxel-2'-hydroxy function by a heterobifunctional poly(ethylene glycol) linker. The resulting conjugate, designated PTXPEGBBN[7-13], was soluble to the upper limit of tested concentrations (250 mg/mL). The conjugate completely retained the receptor binding properties of the attached peptide as compared with those of the unconjugated BBN[7-13]. In experiments with NCI-H1299 human nonsmall cell lung cancer cells, the cytotoxicity of the PTXPEGBBN[7-13] conjugate at a 15 nM dose was enhanced by a factor of 17.3 for 24 h and 10 for 96 h exposure times, relative to paclitaxel. The IC(50) of the conjugate, tested against the same cell line, was lower than the free drug by a factor of 2.5 for both 24 h and 96 h exposures. These results describe, for the first time, the design and synthesis of a soluble tumor-directed paclitaxel prodrug which may establish a new mode for the utilization of this drug in cancer therapy.

Combined modality therapy of A431 human epidermoid cancer using anti-EGFr antibody C225 and radiation.

BACKGROUND: Monoclonal antibodies (mAb) to epidermal growth factor receptor (EGFr) inhibit tumor cell proliferation and enhance cytotoxicity of chemotherapeutic agents. The purpose of this study was to investigate the interaction of the anti-EGFr antibody C225 combined with radiotherapy (RT) on EGFr expressing A431 human epidermoid cancer cells. METHODS: Cell proliferation, apoptosis, EGFr expression and phosphorylation, and clonogenic survival were assayed in vitro. A431 tumor growth inhibition and immunohistochemistry analysis of EGFr expression and apoptosis were assessed in vivo. RESULTS: C225 plus RT produced greater inhibition of A431 cell proliferation than C225 or RT alone which was corroborated by enhanced apoptosis. Similar clonogenic survival occurred following the addition of C225 to RT, although colonies were smaller in the presence of C225. C225 produced inhibition of EGF-induced phosphorylation of EGFr without concurrent down-regulation of surface receptor, which was not altered by RT. Combined treatment of mice bearing tumors demonstrated enhancement of complete regressions, reduction in time to tumor size doubling, and prolongation of survival. Significant apoptosis occurred in xenograft tumors treated with C225 with or without RT. CONCLUSIONS: These data demonstrate an interaction between C225 and RT. C225-mediated apoptosis and inhibition of EGFr phosphorylation may be critical in the interaction. Studies to define the precise influence of combined modality treatment on the EGFr signal transduction cascade need to be pursued. The combination of growth factor receptor antibodies and RT has potential application in clinical oncology.

Constitutive production of a murine retrovirus in the human B-lymphoblastoid cell line, DG-75.

During the screening of human lymphoblastoid cells as suitable hosts for retrovirus transmission studies, the Epstein-Barr virus (EBV)-negative, B-lymphoblastoid cell line DG-75 was found to be chronically infected with a heretofore unrecognized retrovirus. Two DG-75 sublines obtained from different sources (designated UW and KAR) were found to produce constitutively particles identified as retroviral by electron microscopy and reverse transcriptase activity. The ultrastructure, morphogenesis, and density in sucrose of the particles were typical of C-type retroviruses. Immunoblot analysis of the DG-75(UW) retrovirus proteins showed antigenic similarity to Moloney murine leukemia virus. A third DG-75 subline in early passage, designated HAD, was free of retrovirus. The DG-75(UW) retrovirus was infectious and produced progeny virions that could be passaged to uninfected cells. We have thus demonstrated that DG-75 cells, which have been used extensively in studies of the biological effects of EBV-encoded genes and their promoters, may be chronically infected with a murine retrovirus and that an early passage subline is retrovirus free and available for such studies.

Ruthenium red preserves glycoprotein peplomers of C-type retroviruses for transmission electron microscopy.

Peplomers, the glycoprotein projections of the outer viral envelope, are distinctive for many viruses. Peplomers of retroviral C-type particles are fragile and are not preserved in standard preparations for transmission electron microscopy of thin sections, whereas the peplomers of B- and D- type retroviruses are usually preserved. Ruthenium red, extensively used in transmission electron microscopy to enhance the preservation of glycosylated proteins, was used in the preparation of three retrovirus-producing lymphoblastoid cell lines: murine SC-1 cells producing the C-type murine leukemia retrovirus LP-BM5 that causes immunodeficiency, human DG-75 cells producing a murine leukemia retrovirus, and human C5/MJ cells producing human T-cell lymphotropic virus type I (HTLV-I). Fixation of cells was carried out with ruthenium red present in the glutaraldehyde, osmium tetroxide, and the ethanol dehydration through the 70% ethanol step. The detailed structure of peplomers of these three different viruses was well preserved.

A human B-lymphoblastoid cell line constitutively producing Epstein-Barr herpesvirus and JHK retrovirus.

The human B-lymphoblastoid cell line, designated JHK-3, with pre-B-cell characteristics, chronically produces two viruses, Epstein-Barr virus (EBV) and JHK virus, an apparently novel retrovirus. The JHK-3 cells are much more productive of extracellular EBV than the high-producer marmoset line B95-8. The extracellular virus of the JHK-3 EBV strain is relatively fragile, more broadly dispersed in an ultracentrifuged sucrose gradient than the B95-8 EBV and more susceptible to disruption by combined treatment with urea and dithiothreitol. By restriction fragment length polymorphism analysis, the JHK-3 EBV strain resembles the EBV strain FF-41. The JHK-3 cells also produce an incompletely characterized, relatively fragile, enveloped, icosahedral RNA virus that contains Mn(++)-dependent reverse transcriptase. JHK virions measure 85 nm in ultrathin sections, much smaller than other Retroviridae. The JHK virus exhibits a distinctive morphogenesis, most nearly resembling C-type retroviruses. The JHK-3 cell line provides a human cell model for investigating virus/virus interactions and their pathogenetic affects on host cells which chronically and simultaneously produce DNA and RNA viruses.

Expression of somatostatin receptor subtypes in breast carcinoma, carcinoid tumor, and renal cell carcinoma.

The presence of transcripts for somatostatin receptor (SSTR) subtypes 1, 2, 3, and 4 was probed by reverse transcription and polymerase chain reaction in ribonucleic acid isolated from 46 malignant and 9 nonmalignant breast tissues, 15 carcinoid tumor tissues, and 13 renal cell carcinoma tissues. The transcripts for SSTR2 were amplified in all but 2 tissue samples, whereas transcripts for SSTR1, SSTR3, and SSTR4 were detected sporadically. In renal cell tumors, SSTR3 transcripts were completely absent. In breast cancer tissue, SSTR subtypes were transcribed independently of patient age, menstrual status, diagnosis, histological grade, and levels of estrogen receptor and progesterone receptor. The probability of finding transcripts for SSTR subtypes, P, was ranked differently for the three types of tumor tissues. For breast cancer, P2 > P3 = P1 > P4; for carcinoid tumors, P2 > P1 > P3 = P4; and for renal cell tumors, P2 > P1 > P4 > P3.

Intracellular virus-induced polypeptides of pestivirus border disease virus.

Intracellular virus specific polypeptides of pestivirus, border disease virus (BDV) in bovine turbinate cells were analysed by radio-immunoprecipitation with specific antisera. Eleven viral polypeptides with molecular weights of 220, 165, 118, 84, 66, 58, 55, 53, 45, 37 and 31 kDa, respectively, were detected in infected cells. Of these, the 165, 118, 84, 66, 58, 55, 53, 45 and 31 kDa proteins were found to be glycosylated. Comparative studies indicated that the polypeptides induced by BDV share many antigenic epitopes with those of the polypeptides induced by bovine viral diarrhea virus (BVDV), a serologically related virus of the same genus, pestivirus. The polypeptide profile of BDV appeared to be more similar to that of the noncytopathic BVDV strain NY1 compared to that of cytopathic BVDV strains NADL and Singer. Peptide mapping analysis of homologous polypeptides from BVDV and BDV confirmed their structural relatedness.