Original Tumor Tissue Research Articles

Significance of P53 in human thyroid tumors.

Mutational changes in the p53 tumor suppressor gene are the most frequent genetic alterations in human malignant tumors. Studies have shown a correlation of p53 expression in breast cancer with tumor prognosis. In contrast to mutational activation of ras and GSP in thyroid tumors, little is known about the role of p53 in thyroid tumor development. Therefore thyroid tumors and thyroid tumor cell lines were studied for the presence of p53 mutations. Snap-frozen tissues from 57 differentiated thyroid carcinomas (DTCs) and 5 goiters were studied by immunohistochemical methods. A panel of six antibodies (pAb 240, 421, 1620, 1801, DO7, and CM1) was employed by using the ABC technique. Five cell lines from DTCs (FTC133, 236, 238, PTC337, MTC164) were examined by the same technique. Additionally, genomic DNA from the cells was amplified by the polymerase chain reaction (PCR) and the PCR product studied for p53 mutations (R273H) by mutation-specific oligonucleotide hybridization (MOH) and temperature gradient gel electrophoresis (TGGE) for the p53 exon 8. None of the benign thyroid tumors and 7 of 57 (12%) DTCs strongly express p53 with a heterogeneous distribution in the tumor tissue. All seven patients have metastatic disease or dedifferentiated tumors G3 (three of seven). CM1 was positive in two cell lines (FTC-133, PTC-337), questionable in FTC-238, and negative in FTC-236 and MTC-164. All three follicular cell lines, however, and the original tumor tissue showed the same p53 mutation (R273H) in MOH analysis and TGGE. P53 mutations are rare in thyroid tumors, but the presence of p53 mutations indicates a poor prognosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of surviving tumor cells after chemoradiotherapy in tongue and floor of mouth carcinomas.

Conservative surgery according to the clinical response to induction therapy is applied in breast carcinomas but not yet in head and neck carcinomas. The possibility of conservative surgery after induction therapy for preservation of function was examined. Forty-three tongue and 15 floor of mouth carcinomas that had received induction therapy and tumor resection were submitted for a comparative investigation on the clinical effect of induction therapy and cancer cell degeneration, which was examined in semiserial specimens from the bread-loaf sections of the excised materials, and a classification of the surviving tumor cell distribution in the tissue was proposed. The distribution was divided into four patterns according to the dispersion and volume of the remaining tumor cells (pattern I, no surviving tumor cells; pattern IIA, viable tumor cells in the central superficial region of the original tumor tissue; pattern IIB, viable cells in the superficial portion, but distributed widely; pattern III, massive tumor cell nests in the deep portion, but centrally localized; pattern IVA, sporadical viable cell islands, but not in the periphery; and pattern IVB, diffuse viable cells scattered throughout the original tumor tissue). The original tumor size did not affect the remaining viable tumor cell distribution pattern, but sporadic distribution patterns (patterns IVA and IVB) were observed in most of the T4 cases. In many cases, especially in tumors of the nondiffuse invasion type (invasion of grade 1 and 2), the tumor remission rate was inversely correlated with the dispersion of the viable tumor cells. However, some cases of grade 3 and 4 invasion exhibited sporadical surviving tumor cells even though a high tumor remission rate was obtained. These tumors, especially those located on the floor of the mouth, recurred with high frequency. Conservative surgery can be applied to nondiffuse invasion cases, according to the tumor remission rate. In T3 and T4 cases exhibiting grade 3 or 4 invasion, conservative surgery is ill-advised, even if more than 90% tumor remission is obtained.

Morphological appearance, growth kinetics and glial fibrillary acidic protein (GFAP) expression in primary in vitro explant culture of astrocytic neoplasms.

Astrocytomas of different grades of malignancy were cultured as primary explant and their sequential growth pattern, glial fibrillary acidic protein (GFAP) expression and labelling index (LI) using bromodeoxyuridine (BrdU) were assessed and correlated with the grade of malignancy of the original tumor tissue. Low-grade astrocytomas showed patterns of growth that diverged from anaplastic astrocytomas and glioblastoma multiforme. The GFAP expression decreased with increasing time in culture in all astrocytomas irrespective of the grading. Maximum GFAP was, however, expressed in the morphologically well-differentiated stellate cells. Contrary to expectations, lower BrdU LI was observed in glioblastoma multiforme in comparison to low-grade astrocytomas, which suggests some unidentified mechanism of differentiation in vitro for astrocytomas of higher grade of malignancy. Hence, in contrast to reported literature on the prognostic value of studies on primary cultures, the present study cautions the extrapolation of the in vitro findings for astrocytomas.

Establishment of a human chorionic gonadotropin‐producing human gastric carcinoma in nude mice

A human chorionic gonadotropin (HCG)-producing gastric carcinoma was transplanted into BALB/c nu/nu nude mice. The original tumor tissue had been obtained by gastrectomy from a 55-year-old patient with gastric carcinoma. The tumor was transplanted serially in nude mice, and its doubling time was stable to approximately 12 days. A positive correlation was observed between serum HCG level and tumor weight. The serum HCG level of the mice and the histology of the tumor weight. The serum HCG level of the mice and the histology of the tumor, papillary adenocarcinoma with partial poorly differentiated adenocarcinoma, did not change on serial transfers. HCG was positively stained by an immunochemical technique only in the site of the poorly differentiated adenocarcinoma, while staining was negative in the site of the papillary adenocarcinoma, which constituted most of the tumor. These results indicate that this tumor in nude mice will be useful not only as a therapeutic experimental system but also for studying the potential malignancy of HCG-producing cancer cells.

A potent and specific immunotoxin for tumor cells expressing disialoganglioside GD2.

Monoclonal antibody 14G2a (anti-GD2) reacts with cell lines and tumor tissues of neuroectodermal origin that express disialoganglioside GD2. mAb 14G2a was coupled to the ribosome-inactivating plant toxin gelonin with the heterobifunctional cross-linking reagent N-succinimidyl-3(2-pyridyldithio)propionate. The activity of the immunotoxin was assessed by a cell-free translation assay that confirmed the presence of active gelonin coupled to 14G2a. Data from an enzyme-linked immunosorbent assay demonstrated the specificity and immunoreactivity of the 14G2a-gelonin immunotoxin, which was identical to that of native 14G2a. Assays for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) revealed that these functional properties of the native 14G2a antibody were also preserved in the 14G2a-gelonin immunotoxin. The gelonin-14G2a immunotoxin was directly cytotoxic to human melanoma (A375-M and AAB-527) cells and was 1000-fold more active than native gelonin in inhibiting the growth of human melanoma cells in vitro. The augmentation of tumor cell killing of 14G2a-gelonin immunotoxin was examined with several lysosomotropic compounds. Chloroquine and monensin, when combined with 14G2a-gelonin immunotoxin, augmented its cytotoxicity more than 10-fold. Biological response modifiers such as tumor necrosis factor alpha and interferon alpha and chemotherapeutic agents such as cisplatinum and N,N'-bis(2-chloroethyl)-N-nitrosourea (carmustine) augmented the cytotoxicity of 14G2a-gelonin 4- to 5-fold. The results of these studies suggest that 14G2a-gelonin may operate directly by both cytotoxic efforts and indirectly by mediating both ADCC and CDC activity against tumor cells; thus it may prove useful in the future for therapy of human neuroectodermal tumors.

Multicellular tumor spheroids from human gliomas maintained in organ culture

Tumor tissue from seven human gliomas was maintained in long-term agar overlay culture as multicellular organotypic spheroids. Light microscopic and ultrastructural observation of the spheroids displayed morphological features similar to those of the original tumor tissue in vivo; in this respect they were different from spheroids obtained from permanent cell lines. The spheroids contained preserved vessels, connective tissue, and macrophages, revealing a close resemblance to the conditions in the original tumor. Flow cytometric deoxyribonucleic acid measurements of cells from the tumor spheroids and from biopsy material obtained directly from the operation revealed the same ploidy and the same amount of proliferating cells in the spheroids as in the original tumor. Fluorescence microscopy using bromodeoxyuridine (BUdR) incorporation and anti-BUdR monoclonal antibody confirmed the proliferative potential of tumor cells in the spheroids. Diameter measurements showed that the size of the spheroids from two of the tumors increased over time while in three other cases it decreased. Spheroids from the remaining two tumors showed no change in size, even after 80 days in culture. These growth data and the relatively high number of proliferating cells, as measured by flow cytometry, indicate that the degree of cell proliferation and cell loss from the spheroids are closely linked, as is the case for tumors in vivo. The culture system presented provides a valuable alternative to propagation of human tumors in animals.

Human gamma enolase: isolation of a cDNA clone and expression in normal and tumor tissues of human origin.

We have isolated and sequenced a cDNA clone encoding the human gamma enolase. Comparison of our cDNA sequence and the rat gamma enolase sequence revealed 97% homology at the level of amino acid sequence. The two coding regions were 91% homologous on the nucleotide level, whereas the 3' noncoding regions were much less homologous (32%). Further comparison of our cDNA sequence with the human alpha enolase revealed an 82% homology at the amino acid level and a 75% homology at the nucleotide level for the two coding regions, whereas the 3' nontranslated regions were only 30% homologous. Using a portion of the 3' nontranslated region of our cDNA, shown to be specific for human gamma enolase, a single 2.5 kb mRNA was detected in human brain tissue. This same gamma enolase message was also found in a number of human normal nonneuronal tissues, and in several human tumor-derived cell lines. Expression of the mRNA for the gamma enolase subunit should thus be used with caution when identifying the cells of neuronal or neuroendocrine origin.

Neuron-specific gamma-enolase derived from human glioma.

Neuron-specific gamma-enolase in human neurogenic tumors, including gliomas, transplanted gliomas, and permanent human glioma cell lines, was studied quantitatively, using newly established enzyme immunoassay methods, together with immunostaining of the tissue and cell preparations. A significantly high level of gamma-enolase was found in some glioblastomas, astrocytomas and oligodendrogliomas as well as medulloblastomas. Glioblastomas transplanted into mice and cultured cell lines derived from the same origins, as well as the permanent human glioma cell lines, also contained gamma-enolase, although the contents were low compared with findings in the original tumor tissues. Immunohistochemically, gamma-enolase stained intensely in the glioblastomatous cells. Serum gamma-enolase concentrations in some patients with gliomas and those of all the transplanted mice were enhanced. The serum gamma-enolase levels in the mice correlated well with size of the transplanted tumor tissues. These results indicate that neuron-specific gamma-enolase is produced in some neurogenic tumors of nonneuronal origin, therefore, serum gamma-enolase may be a useful biomarker for monitoring the extent of disease in patients with gliomas.

Augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene.

We studied the effect of altered c-myc structure and expression upon the ability of c-myc to promote the transformation of normal rat embryo cells when it was supplemented by EJras (the mutant c-H-ras1 gene from EJ/T24 bladder carcinoma cells). We tested several c-myc alleles cloned from normal and tumor tissues of chicken and human origin and found that only LL4myc (derived from a bursal lymphoma in which an avian leukosis virus long terminal repeat resides within the first c-myc intron in the same transcriptional orientation) had cotransforming activity. No activity was observed with normal chicken and human c-myc alleles, two other bursal lymphoma c-myc alleles (LL3myc and LL6myc), and two human c-myc genes (HSRmyc and DMmyc) from human neuroectodermal tumor cell line COLO320, in which c-myc is amplified. Some of these inactive alleles had the following alterations that are frequently found in tumor-derived c-myc: point mutations affecting the encoded protein (LL3myc); a truncated structure with loss of the first, noncoding exon (LL3myc and DMmyc); and proviral integration within or near the myc locus (LL3myc and LL6myc). The following two experimental approaches indicated that cotransforming activity was directly related to the transcriptional activity of the alleles in cultured rat cells: when cotransfected into Rat-2 cells, LL4myc was more highly expressed than the other (inactive) alleles; and augmented expression of HSRmyc, DMmyc, or normal human or normal chicken c-myc placed under the transcriptional control of retroviral long terminal repeats or increased expression of normal human c-myc under the influence of a retroviral enhancer element was accompanied by cotransformation activity. We concluded that augmented expression of even a normal c-myc gene is sufficient for cotransforming activity and that additional structural alterations frequently found in tumor-derived alleles are neither necessary nor sufficient for the gene to acquire rat embryo cell cotransforming properties.

Augmented expression of normal c-myc is sufficient for cotransformation of rat embryo cells with a mutant ras gene.

We studied the effect of altered c-myc structure and expression upon the ability of c-myc to promote the transformation of normal rat embryo cells when it was supplemented by EJras (the mutant c-H-ras1 gene from EJ/T24 bladder carcinoma cells). We tested several c-myc alleles cloned from normal and tumor tissues of chicken and human origin and found that only LL4myc (derived from a bursal lymphoma in which an avian leukosis virus long terminal repeat resides within the first c-myc intron in the same transcriptional orientation) had cotransforming activity. No activity was observed with normal chicken and human c-myc alleles, two other bursal lymphoma c-myc alleles (LL3myc and LL6myc), and two human c-myc genes (HSRmyc and DMmyc) from human neuroectodermal tumor cell line COLO320, in which c-myc is amplified. Some of these inactive alleles had the following alterations that are frequently found in tumor-derived c-myc: point mutations affecting the encoded protein (LL3myc); a truncated structure with loss of the first, noncoding exon (LL3myc and DMmyc); and proviral integration within or near the myc locus (LL3myc and LL6myc). The following two experimental approaches indicated that cotransforming activity was directly related to the transcriptional activity of the alleles in cultured rat cells: when cotransfected into Rat-2 cells, LL4myc was more highly expressed than the other (inactive) alleles; and augmented expression of HSRmyc, DMmyc, or normal human or normal chicken c-myc placed under the transcriptional control of retroviral long terminal repeats or increased expression of normal human c-myc under the influence of a retroviral enhancer element was accompanied by cotransformation activity. We concluded that augmented expression of even a normal c-myc gene is sufficient for cotransforming activity and that additional structural alterations frequently found in tumor-derived alleles are neither necessary nor sufficient for the gene to acquire rat embryo cell cotransforming properties.

Establishment and characterization of a human glioblastoma cell line with a stable karyotype and nullisomy 13

A permanent cell line (HeRo) with a stable karyotype (80–84,XXYY) and with defined numerical and structural chromosome aberrations was established from a human glioblastoma, a highly malignant brain tumor. Transformation of these cells with SV40 led to a second permanent cell line (HeRo-SV) with a reduced, but also stable, karyotype (72–74,XXYY). The morphological appearance of the glioblastoma line was similar to the main component of the original tumor tissue. The transformed cells differed from their counterparts in accelerated growth, enhanced growth in soft agar, reduced growth conditions, expression of SV40 T antigen, and altered epitheloid morphology. Both cell lines have been grown in continuous culture for more than 2 years. The stability of both the biologic properties and the karyotypic changes induced by SV40 is quite remarkable. Both lines show a nullisomy 13.

骨・軟部しゅようの組織培養

We have used tissue culture techniques chiefly on malignant tumor of bone or soft tissue for these three years. Recently, several interesting findings have been obtained using the methods of tissue culture with electron microscopy and histochemical techniques. This report deals with some of them. Cultured cells revealed the morphological similarity to those of original tumor tissues. Cultured cells of synovial cell sarcoma disclosed the biphasic pattern of spindle cells and epithelial-like elements.In the case of malignant giant cell tumor of soft part, histiocyte like cells and fibroblast like cells were identified by electron microscopy. However the results that this tumor showed the positivity for α-naphthyl acetate esterase suggest the possibility of its histiocytic origin.

Identification of carcinogens by measurement of cell-mediated immunity: II. Assay specificity

Studies utilizing the Fischer F334 inbred rat model were undertaken to define the specificity for antitumor cell-mediated immune (CMI) measurements as determined by lymphoid-cell cytotoxicity to determine whether such values could serve as an analytical reflection for the carcinogenic potential of environmental pollutants. The investigations revealed that specificity was apparently limited to a carcinogenic insult and designated by the embryonic origin of the affected tissue. For example, it was found that animals having X-ray-induced small-bowel, 1,2-dimethylhydrazine-induced colon, and 7,12-dimethylbenz[ a]anthrazene-induced pancreas adenocarcinomas possessed lymphoid cells cytotoxic to tumor target cells of either the small bowel or pancreas, but were not lytic to tumor tissue of ectoderma or mesodermal origin. Nor was injury inflicted upon normal intestinal, kidney, or tail fibroblastic cells, while significant lysis was seen when normal fetal cells were used as targets. The findings also suggest that the presence of the carcinogen in the assay has little influence upon the CMI values. Thus, the present investigation continues to support the concept that immunological responsiveness may serve as a useful measure for specifically identifying and quantitating effects of environmental carcinogens.

Autoradiographic investigation of schirrous carcinoma of the human stomach by culture in diffusion chambers

When a schirrous carcinoma of the human stomach was cultured in diffusion chambers, growth of epithelial and connective-tissue cells was observed. Unlike the original tumor tissue in vitro when the connective-tissue cells did not incorporate thymidine-H3, during growth in diffusion chambers they were labeled. The fraction of cells with labeled nuclei 1 h after injection of thymidine-H3 in the zone of growth averaged 25.1%, much greater than the proportion of labeled cells determined after incubation of the original tumor tissue in vitro (6.6%). Under the conditions of culture, a rapidly proliferating subpopulation of cells with a mitotic cycle 14.8 h in duration was discovered.

Studies on the mechanisms of invasion in cancer. II. In vivo effects of a factor chemotactic for cancer cells.

AbstractA substance chemotactic for tumor cells was isolated from some tumor tissues of animal and human origin and highly purified. It induced an extravascular emigration of circulating tumor cells on experimental injection, but no emigration of polymorphonuclear leukocytes. The emigrated tumor cells proliferated in the extravascular tissue and invaded the surrounding tissues, and a metastatic secondary tumor was formed. This material had no activity to increase vascular permeability. Permeability factors, isolated from tumor tissues, histamine and bradykinin were ineffective for tumor‐cell emigration. A factor chemotactic for polymorphonuclear leukocytes, isolated from inflamed sites, induced an emigration of the cells on experimental injection, but no emigration of tumor cells. These observations suggested that the cancer cell chemotactic factor might be associated with malignant invasion.

Studies on the mechanisms of invasion in cancer. I. Isolation and purification of a factor chemotactic for cancer cells.

AbstractA substance chemotactic for cancer cells was extracted from the pseudoglobulin fraction of some tumor tissues of animal and human origin. It was highly purified by column chromatography using Sephadex G‐50 and CM‐Sephadex and then by disc electrophoresis; it behaved as a homogenous substance on disc electrophoresis. The substance was a protein with a molecular weight of approximately 70,000. It was thermolabile and had no proteolytic activity. The material was similarly active for rat ascites hepatoma AH109A cells, mouse ascites hepatoma MH134 cells and mouse myeloid leukemia C‐1498 cells, suggesting a common chemotactic action. It was ineffective for polymorphonuclear leukocytes of rats. No such chemotactic activity was demonstrated by the protein fraction from normal skin and muscle.

Some characteristics of human cancer tissues in vitro.

Eighty-six tumor tissues of diverse histology and origin were cultured in vitro. Some growth characteristics were studied with the aim of establishing criteria which might help in prognosis. It was found that tumors of identical origin and classification differed in their ability to grow in either cell or tissue cultures and, in the effect of cancer ascitic fluid on their growth and in morphology of the developing cultures. The main group of tumors tested was the invasive duct carcinoma of the breast, in which we found a wide spectrum of morphological variations which ranged from well-organized homogenous cell layers to small colonies of round aggregating cells which detached quickly. These differences will be correlated with the results of the clinical follow-up being done on the respective patients.

ANTIGENICITY OF PAPILLARY SEROUS CYSTADENOCARCINOMA TISSUE CULTURE CELLS

Abstract The antibodies produced by immunization of rabbits with sonicated tissue culture cells of an established strain of human papillary serous cystadenocarcinoma of the ovary were investigated by Ouchterlony double gel diffusion, immunoelectrophoresis, and cytotoxicity studies. Cross-reactivity with normal tissues was eliminated by adsorption of the antisera on normal ovary homogenates. The resulting serum reacted only with the original tissue culture cells and ovarian tumor tissue of the same type; it did not show precipitin lines with any of the normal tissues or other tumors tested. These findings indicate the presence of a specific antigen in the papillary serous cystadenocarcinoma cells.

Growth <italic>In Vitro</italic> and Ultrastructure of Cells From a Medullary Carcinoma of the Human Thyroid Gland: Transformation by Simian Virus 40 and Evidence of Thyrocalcitonin and Prostaglandins

Continuous single layer tissue cultures were established with explants from a metastatic nodule of medullary carcinoma primary in the thyroid gland. Some cultures were infected with simian virus 40 and the cells acquired morphologic and growth characteristics associated with viral transformation. Thyrocalcitonin activity was detected by radioimmunoassay of culture fluids as late as 7 months after initiation of the single layers. Substances with the chemical and biological properties of (PG) prostaglandins E2 and F2alpha were extracted from culture fluids after 4 months. The original carcinoma cells contained abundant cyctoplasmic secretory granules, which were demonstrated by electron microscopy. These granules disappeared during subculture of the monolayers, and electron-dense secretory substance accumulated within dilated cisternae of the ribosome-bound endoplasmic reticulum. Ultrastructural differences in surface organization of the virus transformed and uninfected culture cells were noted. Amyloid was present in the original tumor stroma and tissue culture explants, but could not be identified in those serial subcultures derived from the monolayer outgrowths.

CYTOPATHOGENIC MYCOPLASMAS ASSOCIATED WITH TWO HUMAN TUMORS. I. ISOLATION AND BIOLOGICAL ASPECTS.

Armstrong, D. (The Children's Hospital of Philadelphia, Philadelphia, Pa.), G. Henle, N. L. Somerson, and L. Hayflick. Cytopathogenic mycoplasmas associated with two human tumors. I. Isolation and biological aspects. J. Bacteriol. 90:418-424. 1965.-Mycoplasmas were isolated from cell cultures of two benign human tumors. The first isolate contained two mycoplasmas, one a well-known human species (Mycoplasma hominis type I) and frequent tissue culture contaminant, and the other a recently reported new type. The second isolate was a mycoplasma of the newly described type. The mycoplasmas could be reisolated, after one or more passages through tissue culture, from extracts of the original tumor tissue. The relationship of the organisms to the neoplasms remains obscure. Both isolates produce cytopathic effect (CPE) and acidification of medium in a variety of tissue cultures. The CPE may be diminished, but not abolished, by increasing concentrations of arginine in the tissue culture media. Infection of various tissue cultures with the mycoplasmas did not result in interference to super-infection with vesicular stomatitis virus.