Allogeneic tumor rejection induced by the intravenous injection of Lyt-2+ cytolytic T lymphocyte clones.

The in vivo activity of murine Lyt-2+ cytolytic T lymphocyte clones was assessed in a tumor allograft model system. Mice that had been sublethally irradiated 16 h previously were injected intraperitoneally with 131I-IUdR-labeled tumor cells. Simultaneously, various doses of four cytolytic T cell clones were injected intravenously and the mice monitored for tumor cell elimination by whole-body counting tecniques. These four clones had been selected on the basis of their ability to proliferate in response to alloantigens in the absence of added T cell growth factor(s). With two of the four clones tested, rapid elimination of tumor cells within the peritoneal cavity was observed, as early as 48 h after intravenous injection of the cloned T cells.

Chloramphenicol: effects on mouse myeloma cells in tissue culture.

Within 36 hours of being administered, chloramphenicol (50 micrograms per milliliter) inhibits by 50 percent the rate of protein synthesis in mouse myeloma cells grown in suspension culture. Although there is a decrease in the amount of globulin synthesized, the rate of synthesis per cell is unchanged; the observed decrease is traced to the inhibition of cell proliferation caused by chloramphenicol.

Calcium spike electrogenesis and other electrical activity in continuously cultured small cell carcinoma of the lung.

Spike electrogenesis, local depolarizing and hyperpolarizing responses, spontaneous rhythmic firing, and alternating resting potentials were measured in cells from a continuously cultured small cell carcinoma of the lung. Spike generation was blocked by MnCl2. In view of this evidence for calcium-spike electrogenesis and previous evidence of secretory activity in these cells, this cell line (DMS 53) can provide a model for the study of excitation-secretion behavior in human neoplastic cells.

Monoclonal antibodies reactive with small cell carcinoma of the lung.

Murine monoclonal antibodies (MoAb) reactive with antigens associated with small cell carcinoma of the lung (SCCL) were prepared and partially characterized. Four were selected for further study on the basis of their lack of reactivity with normal leukocytes and erythrocytes. These MoAb, designated SCCL-41, SCCL-114, SCCL-124, and SCCL-175, are all IgM immunoglobulins. The binding of these MoAb to patient-derived SCCL tumor cells, SCCL cell lines, and non-SCCL cell lines was studied by indirect immunofluorescence and flow cytometry. Considerable heterogeneity in the expression of these cell surface antigens was noted among both the patient-derived tumor cells and the SCCL cell lines. One of the MoAb, SCCL-175, reacted with 7 of 7 patient-derived tumor cell samples and 9 of 10 SCCL cell lines. None of the antigens defined by these MoAb were expressed on non-SCCL lung tumor cell lines. SCCL-175 reacted with cells from both a choriocarcinoma and a colon carcinoma cell line, whereas the other 3 MoAb were unreactive with these and several other tumor cell lines. These MoAb may be useful in the diagnosis and subclassification of SCCL tumors.

Karyotypic evolution associated with loss of tumorigenicity.

A reproducible association between loss of tumorigenicity and specific karyotypic changes was described in cell culture lines SLU-5 and DMS-402 established from mouse plasmacytoma MOPC-21 carried in BALB/c mice. The defect in chromosome no. 15, which has been specifically associated with mouse myelomas, was neither corrected nor eliminated in the karyotypic evolution that occurred simultaneously and progressively with the grandual loss of oncogenicity.

Karyotype, marker formation, and oncogenicity in mouse plasmacytomas.

Two common chromosome markers in the 2 plasmacytomas previously examined by Giemsa banding were consistently present in the mouse plasmacytoma X-5563, a transplantable hypertetraploid tumor of spontaneous origin in C3H mice. The 2 markers were found in both induced and spontaneous tumors and in either BALB/c or C3H mice. The derived cell line had 17 fewer chromosomes than the X-5563 tumor and was oncogenic, and its modal karyotype was identical to that of the tumor transmitted by the inoculation of the cell line. The homogeneity of a slight karyotypic modification in a second tumor suggested a possible clonal origin of that tumor. The high frequency of centric fusions between homologues and the structure of certain markers suggests that homologue association may precede marker formation. We proposed a second mechanism of marker formation, selective regional elongation, to account for the larger number of markers with proximal or distal elongations without evidence of translocation and for the observed alterations in length and banding pattern of markers after growth in vitro. Comparison of MOPC-21, MOPC-315, and X-5563 tumors showed preferential involvement of certain chromosomes in marker formation, an inferred association of the 2 common markers with an early stage in the origin of the 3 plasmacytomas, and consistent loss of an X chromosome. Loss of oncogenicity in cell lines was associated with a number of karyotypic changes, but did not require the loss of the characteristic markers or additional copies of a specific normal chromosome.

A specific chromosome breakpoint associated with mouse plasmacytomas.

The chromosomes of uncultured cells of the near-diploid mouse plasmacytoma MOPC-31C were studied. The modal number of chromosomes was 44. The tumor lacked two marker chromosomes, reciprocal translocation [rcp t(12; 15)], that in previous studies were found to be common to 3 other uncultured myelomas and 1 cultured mouse myeloma. Through the formation of two markers, rcp t(6; 15), unique to this tumor, however, the tumor shared with other tumors and their specific markers a common breakpoint in chromosome "15 at band D3/E. This breakpoint has been found in all mouse plasmacytomas examined with banding thus far and is considered of possible importance in the development of this tumor.

Isolation and characterization of a hormone-producing cell line from human small cell anaplastic carcinoma of the lung.

A continuous cell culture line was established from a bone marrow metastasis of small cell anaplastic carcinoma of the lung. The cultures were characterized by light and electron microscopy, and an unusual concentric arrangement of cells was observed, both in sectioned material from the patient's tumor and from the cell cultures. The cells had two types of specialized cell junctions and contained secretory-like granules of the type described in neuroendocrine cells. Lactic dehydrogenase isozyme patterns were the same as those observed in normal human serum, and the karyotype revealed the presence of several marker chromosomes. Vasopressin was present in the cells and secreted into the culture medium in the absence of neurophysin, as shown by the immunoperoxidase technique and radioimmunoassay. Oxytocin was also absent from cells.

Biosynthesis of procalcitonin in small cell carcinoma of the lung.

Immunoreactive calcitonin (CT) secreted by DMS 53, a cell line derived from human small cell carcinoma of the lung, consists almost entirely of molecular species larger than the mature hormone (Mr 3,420). Messenger RNA isolated from DMS 53 cells and nude mouse tumors was translated in wheat germ systems, and the products were precipitated with CT-specific antisera. Analyses of the translation products by electrophoresis on 15% polyacrylamide-sodium dodecyl sulfate gels indicated synthesis of a Mr 16,500 preprohormone that was reduced to Mr 14,500 by cotranslation with microsomal membranes. Immunoprecipitation of CT from media from pulse-labeled cultures revealed two major products (Mr 16,500 and Mr 14,500) and up to three minor secreted polypeptides (Mr 9,400, 8,400, and 6,800). Intracellular CT from cell homogenates appeared almost entirely as a single major product (Mr 14,500) and possibly 3-4 minor components (Mr 16,500; 9,200, 8,400, and 6,800). No glycosylated forms of CT were demonstrable by lectin binding methods or labeling attempts with tritiated sugars. The presence of multiple CT species in DMS 53 cells suggests significant post-translational processing of the larger precursor molecules and the accumulation and secretion by small cell carcinoma of the lung of several intermediate immunoreactive forms via a glycosylation-independent secretory pathway.

Occurrence of fibrin and tissue factor antigen in human small cell carcinoma of the lung.

Fibrin was detected by specific immunofluorescence in tissue obtained from five of six cases of small cell carcinoma of the lung. Dense specific fluorescence was observed in the connective tissue stroma surrounding metastatic tumor nodules and frequently in the scant extracellular stroma surrounding individual viable tumor cells and small tumor cell clusters. When observed by electron microscopy, the fibrin hugged tumor cell plasma membranes and, in some areas, seemed to envelop the cells. Fluorescent staining of tumor cells, but not the stroma, was observed with an antibody to tissue factor. These findings suggest that local activation of coagulation occurs in small cell carcinoma of the lung. Deposited fibrin may contribute to the growth and spread of this particular type of cancer.

Expression of myeloid and major histocompatibility antigens on small cell carcinoma of the lung cell lines analyzed by cytofluorography: modulation by gamma-interferon.

Recent reports have described the expression of myeloid cell surface antigens on cells of small cell carcinoma of the lung (SCCL). In order to confirm and extend these findings, we have examined by cytofluorography a large panel of well-characterized cell lines derived from SCCL tumors for the expression of several myeloid cell-associated antigens, some of which were not examined in previous reports. In addition, we have studied the expression of Classes I and II HLA antigens on these SCCL cell lines. Finally, we examined the effect of gamma-interferon on the expression of several cell surface markers and on proliferation of SCCL cells. We have found that several SCCL cell lines expressed a Mr 55,000 polypeptide antigen, My23, previously found only on monocytes and monocytic leukemia cells. In addition, the cell lines studied expressed another antigen, defined by monoclonal antibody AML-1-99, which is associated with monocytes and hematopoietic stem cells. We confirmed previous studies that the Leu-7 antigenic determinant is expressed on SCCL cells but observed only minimal or absent binding of monoclonal antibody OKM1 to most cell lines. Class I HLA antigens were present on eight of nine lines examined while Class II HLA was expressed on three of nine lines. Gamma-Interferon decreased the proliferative rate of all lines examined. However, this lymphokine was capable of inducing Class I HLA on several lines. The effect of gamma-interferon on other cell surface antigens was variable. These studies confirm that some myeloid cell-associated antigens are expressed on cultured SCCL lines and, additionally, show that their expression can be modulated by immune interferon. Determining the significance of finding myeloid cell-associated antigens on SCCL cells will require further study.

Abnormalities in structure and expression of the retinoblastoma gene in small cell lung cancer cell lines and xenografts in nude mice.

The putative retinoblastoma gene (Rb) is a tumor suppressor gene which is believed to cause retinoblastomas when both alleles are inactivated, leading to lack of the encoded Mr 110,000-116,000 phosphoprotein. Inactivation of the Rb gene has also been found in several other tumor types, including small cell lung cancer (SCLC). Absence of the 4.7 kilobase mRNA has been found to be frequent in SCLC, and it has been reported that the Rb Mr 110,000-116,000 protein product is always absent, even in tumors expressing Rb mRNA. Using Western blotting technique with a monoclonal antibody directed against the Rb protein, we investigated the expression of the Mr 110,000-116,000 Rb protein in SCLC tumors grown as xenografts in nude mice and/or as cell lines. Rb messenger RNA expression was determined by Northern blotting, and gross structural gene alterations were investigated by Southern blotting. Tumors established from 23 patients were studied. Seven of the tumors did not express Rb protein, whereas expression was detectable in 13. Three tumors were not investigated for protein expression. Only two tumors expressed Rb mRNA without detectable Rb protein expression. Gross DNA alterations were found in four tumors, of which only one expressed Rb mRNA. Our results demonstrated frequent absence of Rb mRNA and protein in SCLC, but apparently normal Rb mRNA and protein were both expressed in more than one-half of the tumors.

Calcitonin as an indicator of the response of human small cell carcinoma of the lung cells to drugs and radiation.

A calcitonin (CT)-producing cell line (DMS53) established from human small cell carcinoma of the lung was grown as three-dimensional multicellular spheroids in spinner culture or on agar in multiwells, and as tumors in nude (athymic) mice. CT release into the media was directly proportional to spheroid volume. The response of these cells following exposures to X-irradiation, Adriamycin, or diazoacetylcholine iodide was assessed by monitoring levels of CT released into the media by individual spheroids. Levels of CT in the blood of nude mice bearing DMS53 xenografts were directly proportional to tumor volume and decreased proportionally with tumor response to X-irradiation and cisplatin treatment. These results suggest that the DMS53 spheroid and xenograft models may be useful systems to monitor responses to therapy utilizing CT as an indicator of tumor burden.

Detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer.

Mutated KRAS2 commonly can be detected in the plasma/serum of patients with pancreatic or colorectal cancers possessing this mutated gene. Positive assays are more common in patients with higher stage tumors but some smaller cancers can also be detected; occasionally, patients with large tumors have negative assays. Because relatively few patients with low-stage tumors have been evaluated, more studies in patients with smaller tumors are needed to further define the clinical usefulness of these assays. The reasons for variable results, particularly in patients with larger tumors, is unclear, although a variety of factors may be involved. More sensitive assays need to be developed that will increase the detection rates, although the problem of producing false positives must be minimized. The presence of mutated KRAS2 sequences in the plasma/serum seems to be quite specifically associated with the presence of cancer containing this mutated gene. This is an important feature of KRAS2 as a tumor marker. Preliminary studies in patients with pancreatic cancer suggest that assays for mutated KRAS2 can complement the commonly used CA19-9 assay and provide additional clinically useful information. The results from currently completed studies on the detection of mutated KRAS2 in patients with colorectal and pancreatic cancer are promising, and the potential usefulness of KRAS2 as a clinically important tumor marker should encourage future research.

In vitro and in vivo alterations of long-term murine plasmacytoma cultures.

Mouse myeloma cell line (SLU-5) that has undergone lengthy propagation in vitro and become nononcogenic was compared with an earlier oncogenic passage of cells. Immunogenicity of cells cultured for various periods of time was compared with that of the original tumor (MOPC-21). The nononcogenic cells were most immunogenic. Cells that were nononcogenic in normal mice produced tumors in nonlethally irradiated mice. Clones isolated from oncogenic and nononcogenic populations varied widely with respect to ability to produce tumors in mice and to specific globulin production.

Detection of tumor-derived DNA in cerebrospinal fluid.

The sensitivity of PCR-based methods for the detection of DNA offers opportunities for tumor diagnosis from the small amounts of tumor-derived DNA released into body fluids. We report the detection of tumor DNA in the cerebrospinal fluid (CSF) of two patients with intracranial neoplasms. One patient had a metastatic breast carcinoma which contained amplified HER-2/neu genes, and amplified HER-2/neu gene sequences were present in her CSF. The other patient had a glioblastoma which contained amplified epidermal growth factor receptor (EGFR) genes, and amplified EGFR gene sequences were present in her CSF. This report demonstrates that CSF sometimes contains tumor-derived DNA and suggests that PCR examination of CSF DNA may be diagnostically useful.

Ectopic production of somatostatin-like immuno- and bioactivity by cultured human pulmonary small cell carcinoma.

Eleven continuous cultures of human pulmonary small cell carcinoma cells were examined, and eight were shown to secrete quantities of somatostatin-like immunoreactivity (SRIF-LI) ranging from 0.07-27 ng/ml culture medium/4 days, SRIF-LI was also found in a 2-N acetic acid extract of one of three human pulmonary small cell carcinomas obtained at autopsy as well as in the extract of a solid tumor resulting from inoculation of nude, athymic mice with SRIF-LI-producing, cultured small cell carcinoma cells. The SRIF-LI produced by one continuous cell line, DMS 53, was characterized in terms of its immunological, chromatographic, and biological properties. SRIF-LI from DMS 53 culture media and lysed cells was heat stable and exhibited parallel displacement to synthetic SRIF standard in a double antibody RIA. DMS 53 SRIF-LI was quantitatively retained on an immunoaffinity column of sheep anti-SRIF-Sepharose 4B under neutral conditions and could be eluted with 2 N acetic acid. Gel filtration chromatography of immunoaffinity-purified SRIF-LI revealed multiple molecular weight forms, the largest of which had an apparent molecular weight of 10,000-12,000 daltons and may represent a precursor form. This high molecular weight SRIF-LI form was resistant to exposure to denaturing conditions (8 M urea or 4 M urea plus 0.5% mercaptoethanol), suggesting the absence of noncovalent and/or disulfide linkages. A low molecular weight form coeluted with synthetic SRIF. Additional evidence for the identity of this form with the tetradecapeptide was provided by highly specific reverse phase high performance liquid chromatography. The rate of degradation of high molecular weight SRIF-LI by the cultures was markedly reduced in comparison to that of the SRIF monomer, resulting in a preferential accumulation of high molecular weight SRIF-LI in 4-day culture medium. Bioactivity of DMS 53 SRIF-LI was assessed in 4-day primary monolayer cultures of rat adenohypophyseal cells where 10(-10)-10(-9) M synthetic SRIF elicited a linear log-dose suppression of 5 X 10(-4) M synthetic SRIF elicited a linear log-dose suppression of 5 X 10(-4) M dibutyryl cAMP-stimulated rat GH release. Immunoaffinity-purified SRIF-LI from DMS 53 lysed cells and 1-h serum-free incubation medium, which consisted predominantly of monomeric SRIF, was equipotent to synthetic SRIF, SRIF-LI from 4-day culture medium consisted mostly of the high molecular weight form and exhibited a reduced bioassay potency ratio relative to synthetic SRIF of 0.73 (95% confidence limits, 0.99-0.53). Chromatographically purified high molecular weight SRIF-LI had significant bioactivity with a bioassay to immunoassay ratio of 0.19 (95% confidence limits, 0.33-0.09). The demonstration of ectopic SRIF, production by human pulmonary small cell carcinoma is consistent with the proposed derivation of this tumor from a cell type in the amine precursor uptake and decarboxylation cell series.

Ectopic production of high molecular weight calcitonin and corticotropin by human small cell carcinoma cells in tissue culture: evidence for separate precursors.

The DMS-79 continuous line of human small cell lung carcinoma cells, which produces immunoreactive (IR)-corticotropin (ACTH), -lipotropin (LPH), and -beta-endorphin (beta END), was found to produce IR-calcitonin (CT). Two major high molecular weight (HMW) forms of IR-CT were observed after gel exclusion chromatography under denaturing conditions (mol wt. approximately 7,000 and approximately 14,000), as well as a minor HMW IR-CT component (mol. wt. approximately 70,000). None of these IR-CT materials was extracted from DMS-79 medium by affinity chromatography using an ACTH antibody covalently bound to agarose. These results demonstrate ectopic production of HMW forms of CT and ACTH/LPH/beta END by human lung tumor cells in tissue culture, but do not support the existence of a common CT/ACTH/LPH/beta END precursor molecule.

Soluble normal and mutated DNA sequences from single-copy genes in human blood.

Healthy individuals have soluble (extracellular) DNA in their blood, and increased amounts are present in cancer patients. Here we report the detection of specific sequences of the cystic fibrosis and K-ras genes in plasma DNA from normal donors by amplification with the polymerase chain reaction. In addition, mutated K-ras sequences are identified by polymerase chain reaction utilizing allele-specific primers in the plasma or serum from three patients with pancreatic carcinoma that contain mutated K-ras genes. The mutations are confirmed by direct sequencing. These results indicate that sequences of single-copy genes can be identified in normal plasma and that the sequences of mutated oncogenes can be detected and identified with allele-specific amplification by polymerase chain reaction in plasma or serum from patients with malignant tumors containing identical mutated genes. Mutated oncogenes in plasma and serum may represent tumor markers that could be useful for diagnosis, determining response to treatment, and predicting prognosis.

Unlabeled antibody methods in electron microscopy: a comparison of single and multistep procedures using colloidal gold.

A comparative study of five unlabeled antibody methods was conducted on the electron microscopic level using bridging techniques and colloidal gold. The study was based on the principles of the single-step colloidal gold (GLAD) method (Larsson L: Nature 282:743, 1979) and the multistep single- and double-bridge techniques used in postembedding immunoperoxidase procedures (PAP) (Sternberger LA: Immunocytochemistry, 2nd ed. Wiley, New York, 1979). Using medullary thyroid carcinoma and the same lot of primary antiserum (goat anti-calcitonin) for each procedure, it was shown that adequate localization of calcitonin with the single-step GLAD method was attainable only at dilutions of 1:100 or lower. The single-bridge technique using goat anti-calcitonin, sheep anti-goat immunoglobulin (Ig)G, and goat anti-calcitonin and antigen-coated gold, respectively, worked well at dilutions of up to 1:5000 but not at dilutions of 1:10,000, while single- and double-bridging techniques utilizing goat anti-calcitonin, sheep (Sh) anti-goat IgG, and sheep anti-goat IgG-coated gold produced good localization at a 1:10,000 dilution of primary antiserum. A two-step method using goat anti-calcitonin and sheep anti-goat IgG-coated gold, respectively, appeared to be the most sensitive technique, with adequate antigen localization occurring at a dilution of 1:25,000. While in our hands the two-step method appeared superior in sensitivity to the single-bridge IgG-coated gold technique, each method has its own advantages depending on the individual needs of the researcher.