Association of the epidermal growth factor receptor kinase with the detergent-insoluble cytoskeleton of A431 cells.

The epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma cells, A431, was found to be predominantly associated with the detergent-insoluble cytoskeleton, where it retained both a functional ligand-binding domain and an intrinsic tyrosine kinase activity. The EGF-R was constitutively associated with the A431 cytoskeleton; this association was not a consequence of adventitious binding. The EGF-R was associated with cytoskeletal elements both at the cell surface, within intracellular vesicles mediating the internalization of the hormone-receptor complex, and within lysosomes. The EGF-R became more stably associated with cytoskeletal elements after its internalization. The cytoskeletal association of the EGF-R was partially disrupted on suspension of adherent cells, indicating that alteration of cellular morphology influences the structural association of the EGF-R, and that the EGF-R is not intrinsically insoluble. Cytoskeletons prepared from EGF-treated A431 cells, when incubated with gamma-32P-ATP, demonstrated enhanced autophosphorylation of the EGF-R in situ as well as the phosphorylation of several high molecular weight proteins. In this system, phosphorylation occurs between immobilized kinase and substrate. The EGF-R and several high molecular weight cytoskeletal proteins were phosphorylated on tyrosine residues; two of the latter proteins were phosphorylated transiently as a consequence of EGF action, suggesting that EGF caused the active redistribution of the protein substrates relative to protein kinases. The ability of EGF to stimulate protein phosphorylation in situ required treatment of intact cells at physiological temperatures; addition of EGF directly to cytoskeletons had no effect. These data suggest that the structural association of the EGF-R may play a role in cellular processing of the hormone, as well as in regulation of the EGF-R kinase activity and in specifying its cellular substrates.

Regulation of major acute-phase plasma proteins by hepatocyte-stimulating factors of human squamous carcinoma cells.

Human squamous carcinoma (COLO-16) cells release factors which specifically stimulate the synthesis of major acute-phase plasma proteins in human and rodent hepatic cells. Anion exchange, hydroxyapatite, lectin, and gel chromatography of conditioned medium of COLO-16 cells result in separation into three distinct forms of hepatocyte-stimulating factors (designated HSF-I, HSF-II, and HSF-III) with apparent molecular weights of 30,000, 50,000 and 70,000, respectively. None of the preparations contains detectable amounts of thymocyte-stimulating activity. Each of the three HSF forms stimulates the accumulation of mRNA for alpha 1-antichymotrypsin in the human hepatoma cell line, HepG2. When the same factors were added to primary cultures of adult rat hepatocytes, the expression of the same set of plasma proteins was modulated as by nonfractionated medium. The hormonally induced accumulation of mRNA for acute phase proteins is qualitatively comparable to that occurring in the liver of inflamed rats. Unlike in human cells, in rat liver cells dexamethasone acts additively and synergistically with HSFs. The only functional difference between the three HSF forms lies in the level of maximal stimulation. HSF-I represents the predominant form produced by normal human keratinocytes and closely resembles in molecular size and isoelectric point the activity produced by activated peripheral blood monocytes while the larger molecular weight forms are more prevalent in human as well as mouse squamous carcinoma cells. The observation that HSFs from different sources elicit essentially the same pleiotropic response in hepatic cells led to the hypothesis that the species-specific reaction of adult liver cells to inflammatory stimuli is pre-programmed and that the function of any HSF is to trigger and tune the execution of this fixed cellular process.

High levels of intracellular bombesin characterize human small-cell lung carcinoma.

"Small cells" or "oat cells" characterize a virulent form of lung cancer and share many biochemical properties with peptide-secreting neurones. The neuropeptide bombesin is present in all small-cell lines examined, but not in other lung cancer cell lines, suggesting that bombesinergic precursor cells in lung may give rise to this disease.

Localization of integrin receptors for fibronectin, collagen, and laminin in human skin. Variable expression in basal and squamous cell carcinomas.

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Production of a low molecular weight eosinophil polymorphonuclear leukocyte chemotactic factor by anaplastic squamous cell carcinomas of human lung.

A peptide of approximately 300-400 daltons exhibiting in vitro chemotactic activity for human polymorphonuclear (PMN) leukocytes, with a preference for the eosinophil series, was isolated from extracts of anaplastic lung carcinomas of the large squamous cell type obtained from three patients with marked peripheral blood hypereosinophilia and eosinophilic infiltration of the tumors and surrounding normal pulmonary tissues. This chemotactic factor was termed ECF-LSC (eosinophil chemotactic factor of lung squamous cell carcinoma). ECF-LSC appeared in the urine of two of the patients in increasing quantities late in the course of their disease and was also elaborated by long-term cultures of dispersed tumor cells from the same two patients. Three anaplastic large cell bronchogenic carcinomas which were not associated with tumor tissue or peripheral blood eosinophilia, a bronchogenic adenocarcinoma from a patient with only peripheral eosinophilia, and a renal cell carcinoma metastatic to the lungs and associated with transient pleural tissue and fluid eosinophilia were all devoid of ECF-LSC. ECF-LSC from tumor tissue extracts, urine, and tumor cell culture medium was comparable to the mast cell-associated tetrapeptides of the eosinophil chemotactic factor of anaphylaxis (ECF-A) in size, but eluted from Dowex-1 at pH 5.0-3.5 in contrast to the more acidic ECF-A tetrapeptides which eluted at pH 3.2-2.2 ECF-LSC, like the tetrapeptides of ECF-A, had a secondary chemotactic activity for neutrophil PMN leukocytes, but not mononuclear leukocytes, and deactivated both eosinophil and neutrophil PMN leukocytes so that they would not respond to a subsequent in vitro chemotactic stimulus. Eosinophils from the two patients with urinary excretion of ECF-LSC and the highest concentrations in tumor extracts were hyporesponsive in vitro to homologous and heterologous chemotactic stimuli, suggesting that ECF-LSC had deactivated the eosinophils in vivo.

Epidermal growth factor receptor expression in human lung carcinomas defined by a monoclonal antibody.

Acetone-fixed frozen tissue sections from 56 cases of human lung carcinoma were tested for reactivity by an indirect immunoperoxidase technique with a monoclonal antibody (MAb 528) specific for the external domain of the epidermal growth factor receptor (EGFR). MAb 528 reacted with all epidermoid (22/22) and large-cell (4/4) lung carcinomas evaluated. The antibody was also positive with a subset of lung adenocarcinomas (13/21) and did not react with small-cell lung cancers (SCLCs) (0/9). MAb 528 also stained normal bronchial epithelium identified within the tumor sections of 5 cases. Thus EGFR was expressed by all epidermoid and large-cell lung carcinomas examined, a subset of lung adenocarcinomas, and normal bronchial epithelium. EGFR expression was not identified in any of the SCLCs tested. These data imply that immunohistochemical detection of EGFR expression may find future application in distinguishing epidermoid, large-cell, and some adenocarcinomas of the lung from SCLCs.

Establishment and characterization of a human colony-stimulating factor-producing cell line from a squamous cell carcinoma of the thyroid gland.

A human colony-stimulating factor (CSF)-producing cell line, T3M-5, has been established in vitro from a squamous cell carcinoma of the thyroid gland transplanted into athymic nude mice [congenitally athymic BALB/c (nu/nu) mice; Central Institute for Experimental Animals, Kawasaki, Japan]. Contaminating fibroblasts derived from a host nude mouse were eliminated by treatment with antiserum raised against nude mouse cells. T3M-5 cells have been continuously propagated during 3 years. The cells grew in a monolayered sheet with about 22 hours of population-doubling time and showed about 40% plating efficiency. The cells exhibited an epithelium-like morphology resembling the structure of the original tumor and showed tumor takes when inoculated into nude mice. Chromosome analysis revealed the cell line to be a human aneuploid line with a hypertriploid mode. The cells possessed the characteristic function of human CSF production in vitro and produced marked neutrophilia in tumor-bearing nude mice that were inoculated with the cultured cells. The molecular weight of the CSF was estimated at about 27,000 and was stable over the pH range 1.0-9.0 at 4 degrees C for 21 hours. The CSF activity was destroyed by either trypsin or chymotrypsin, but it resisted neuraminidase, DNase, and RNase. The cells could be well propagated in roller bottles. About 100 liters of the conditioned medium was obtained with the roller bottle culture method, which formed approximately 500,000,000 colonies of human bone marrow cells. The rate of recovery of CSF activity from the gel-filtration column was high (68.9%). This cell line is therefore expected to aid in the large-scale preparation of human CSF.

Lectin-binding sites in preneoplastic and neoplastic lesions of human and rodent respiratory tracts.

The localization of fluorescein-labeled lectins, i.e., concanavalin A (Con A), Ricinus communis-120 (RCA), and wheat-germ agglutinin (WGA), were studied histologically in F344 rat epithelial lesions produced in the course of chemical carcinogenesis. WGA could not be demonstrated in these lesions. Although all lesions showed positive-binding sites when high concentrations of either Con A or RCA were used, a dilution study showed that the epithelial lesions had different affinities for lectins. With both Con A and RCA, dysplastic and neoplastic lesions showed the strongest intensity of fluorescence and squamous metaplasia showed the weakest. Normal and hyperplastic epithelia showed intermediate intensity. In the dilution study, RCA showed eight times more affinity and Con A showed two times more affinity for dysplastic and neoplastic epithelia than for normal or hyperplastic epithelium. Similar affinity patterns were observed in human lesions and tumors. With Con A, 58% of tumors showed much stronger fluorescence than did normal epithelium, and 44% of the tumors showed positive fluorescence with RCA. Although both lectins exhibited a stronger affinity for all the dysplastic-neoplastic lesions than for normal or hyperplastic epithelium, RCA proved to be the most adequate marker for preneoplastic lesions.

Antibodies to Herpes simplex virus in Jewish women with cervical cancer and in healthy Jewish women of Israel.

Herpes simplex virus (HSV) antibody titers were examined in sera from 39 Jewish women with squamous cell carcinoma of the uterine cervix (CaCx) and in sera from controls matched by age and country of origin. Highly significant differences were found between the cases and controls for both HSV type 1 (HSV-1) and HSV type 2 (HSV-2). Compared to findings in other demographic areas, the geometric mean titer (GMT) of HSV-1 among the CaCx cases were considerably higher, whereas the GMT for HSV-2 was in the same range. The percentage of HSV-2-positive patients, as defined by the HSV-2/HSV-1 antibody titer ratio was low compared to that found in other demographic areas; this was presumably due to the high incidence of HSV-1 infection in the population. The HSV-1 and HSV-2 infection rate in the Israeli Jewish female population was estimated by antibody titers for 94 healthy subjects. The GMT of HSV-1 was considerably higher, whereas the GMT of HSV-2 was lower, than those reported elsewhere. The association found previously between HSV-2 and CaCx remained true for Jewish women. Their low incidence of CaCx, which did not seem to result from lower susceptibility, might be explained by the low incidence of HSV-2 infection.

Characteristics of four new human cell lines derived from squamous cell carcinomas of the head and neck.

Four human cell lines were established from biopsy specimens of squamous cell carcinomas of the larynx (TR131 and TR138), tongue (TR126), and buccal mucosa that had infiltrated a lymph node (TR146). All 4 lines readily formed colonies on a plastic substratum, but they were virtually incapable of forming colonies in an anchorage-independent semisolid support system of soft agar (cloning efficiencies, less than 0.02%). The proliferation of this group of tumor-derived cell lines, therefore, appeared to be highly anchorage dependent. Keratin filaments could be visualized in each line by indirect immunofluorescence with the use of polyclonal or monoclonal antibodies to keratins; staining with monospecific antibodies indicated that 3 of the 4 lines expressed simple epithelial keratins 8 and 18, whereas 1 of the 4 also expressed keratin 19. A panel of lectins revealed characteristic localization patterns distinct from those observed on other epithelial cell lines. Cells from 3 lines (TR131, TR138, and TR146) inoculated into nude mice (nu/nu) produced cystic nodules or unequivocal tumors having a histology indicating a squamous cell origin for the injected cells. Electron microscopy demonstrated that the cell lines covered a spectrum of differentiation capability ranging from the undifferentiated monolayer cultures of TR126 to the rather well differentiated, stratified cultures of TR131.

Examination of oral cancer tissue for the presence of the proteins ICP 4, ICP 5, ICP 6, ICP 8, and gB of herpes simplex virus type 1.

Although patients with oral cancer have increased levels of antibody to herpes simplex virus type 1, the origin of the antigenic stimulation remains unknown. We have therefore looked for proteins of herpes simplex in oral squamous cell carcinomas by staining frozen sections with monoclonal antibodies to the proteins ICP 4, ICP 5, ICP 6, ICP 8, and gB. No staining was seen of the tumor cells of any of 11 oral cancer cases or of the epithelium of 29 other oral lesions, which included cases of leukoplakia, lichen planus, and aphthous ulcers. Frequent staining of mast cells was seen in the connective tissue associated with oral cancer when ascitic fluid was used as the source of monoclonal antibody, but such staining was not seen when the precipitated IgG fraction was used.

Production of ring-shaped particles by normal and metaplastic tissue. I. Human skin.

Maintenance media incubated with biopsy specimens of human skin tissues contained minute (10-12 nm wide), ring-shaped particles (RSP) similar to those described previously in culture media of mammalian cell lines. In addition to the qualitative demonstration of the particles by electron microscopy, a quantitative method was applied to estimate in media the amount of DNA that could be attributed primarily to RSP content. The amounts of DNA, obtained with 146 test specimens, varied with the pathologic condition of the tissue in the following ascending order: normal skin, verruca vulgaris, seborrheic verruca, actinic keratosis, basal cell carcinoma, and squamous cell carcinoma.

Keratin expression in normal esophageal epithelium and squamous cell carcinoma of the esophagus.

The 8-nm keratin filament is a major component of the cytoskeleton of epithelial cells and epithelial-derived cancers (carcinomas). Recently, it has been shown that the pattern of keratins produced by an esophageal epithelial cell undergoes change upon malignant transformation. In order to evaluate the potential importance of these differences in providing improved diagnostic techniques for pathology, we have investigated the consistency of the patterns of keratins expressed in normal esophageal epithelium, squamous cell carcinoma (SQCC) of the esophagus, and cultured esophageal epithelial cells. In six patients, the keratin pattern expressed by SQCC of the esophagus and corresponding normal esophageal epithelium was consistently different as judged by immunoblot analysis of electrophoretically separated protein extracts. Whereas the SQCCs typically expressed major keratins with molecular weights of 58,000, 56,000, 50,000, and 46,000, the normal esophageal epithelium produced two major keratins with molecular weights of 58,000 and 52,000 and a minor keratin with a molecular weight of 56,000. When normal esophageal epithelial cells were grown in tissue culture, their keratin pattern changed, and keratins with molecular weights of 58,000, 56,000, 52,000, 50,000, 46,000, and 40,000 were expressed. Although some minor variations in keratin patterns were seen, the major differences in keratin pattern expressed by normal esophageal epithelial tissue, SQCC of the esophagus, and cultured esophageal cells were consistent and reproducible.

Changes in glycosphingolipids accompanying the differentiation of human squamous SQCC/Y1 cells.

SqCC/Y1 cells grow as a monolayer in culture and differentiate when maintained in the plateau phase; in the absence of serum these cells differentiate more rapidly. The differentiation is characterized by the stratification of the culture to form a structure consisting of several cellular layers, synthesis of specific keratins, and the attainment of the capacity to form a cornified cell membrane. The stratification process is indicative of the importance of cell-cell interactions during maturation. To study the relationship between membrane glycosphingolipids (GSLs) and the state of differentiation of SqCC/Y1 cells, GSLs were measured in cultures grown in the presence or absence of fetal calf serum. Glycolipids were isolated by diethylaminoethyl-Sephadex and Iatrobeads column chromatographies, and their distributions were determined by high-performance thin-layer chromatography. GM3 was the major ganglioside present in these cells. Other ganglioside components were tentatively identified as GM2, GM1, and GD3. Differences in ganglioside patterns were observed in differentiated cultures; the major changes were accumulation of GD3 and depletion of GM1. The predominant neutral GSLs in SqCC/Y1 cells were identified as Glc beta 1-1Cer, Gal beta 1-4Glc beta 1-1Cer, Gal beta 1-4Gal alpha 1-4Glc beta 1-1Cer, Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, and three unknown complex GSLs. Differentiated cultures, however, showed variations in banding patterns, which include an increase in Glc beta 1-1Cer and Gal beta 1-4Glc beta 1-1Cer and a decrease in Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer and Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta-1Cer. These changes, however, were not observed when the cells were grown in the presence of epidermal growth factor or retinoic acid, factors which inhibit the differentiation process. The findings demonstrate significant changes in glycolipid composition of differentiated SqCC/Y1 cells grown in the absence of serum, suggesting that these lipids may be important to the differentiated state.

Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture.

The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.

Neopterin as a prognostic indicator in patients with carcinoma of the uterine cervix.

In vitro, neopterin, a pyrazinopyrimidine compound, is excreted by human monocytes-macrophages after induction by supernatants from activated T-lymphocytes or by recombinant gamma-interferon. In vivo, it represents a noninvasive test for activation of cellular immune reactions. To evaluate the prognostic value of pretherapeutic urinary neopterin levels and of serial neopterin measurements during follow-up in women with cervical cancer, 1088 urine specimens from 186 consecutive patients were analyzed. Clinical assessments were made without knowledge of the results of neopterin assays (a "blinded" assessment). During the observation period (June 1980 to March 1984), 27 relapses, 18 metastases, and 26 deaths were seen. The prognostic significance of pretherapeutic neopterin and other possible prognostic clinical and laboratory parameters was tested by the univariate and multivariate Cox proportional hazards model using a stratification according to stage and surgical treatment. The combination of age at diagnosis, pretherapeutical hemoglobin, leukocyte count, and neopterin was found to predict survival best. On the basis of this result, risk groups were identified exhibiting markedly different survival behavior. A highly significant association was found between serial neopterin measurements and the risk for a relapse, metastasis, or death. The data suggest that urinary neopterin levels might be a useful adjuvant parameter in monitoring women with cervical cancer.

Glycoproteins distinguishing non-small cell from small cell human lung carcinoma recognized by monoclonal antibody 43-9F.

Cell lines derived from human squamous lung carcinoma release large amounts of a soluble glycoprotein into the culture media, having very high molecular weight (greater than 2 X 10(6] and mucin-like properties. A monoclonal antibody called 43-9F has been generated that recognizes a carbohydrate epitope on the glycoconjugate. The epitope is also present on a diverse set of smaller glycoproteins (Mr 50,000-200,000) distributed primarily on the surface of the squamous lung carcinoma cells. A sensitive assay using the 43-9F antibody in a dot blot procedure has been devised that is able to detect an amount of antigen less than that possessed by a single squamous lung carcinoma cell. This assay, and also conventional immunofluorescence and immunohistochemical assay procedures, have been used to screen different normal cells, normal tissues, cancer cells, and tumor biopsy specimens for the antigen. In the normal lung the 43-9F antigen is found only on cells of some of the seromucous glands. In the normal digestive system it is associated in certain organs only with a limited population of mucosal epithelial cells. Other organ systems lack any reactive cells. The cells of most human non-small cell lung carcinomas and their released glycoconjugates have large amounts of the 43-9F epitope, while small cell lung carcinomas and the glycoconjugates released by small cell lung cancer cells lack the epitope. The oligosaccharide recognized by the 43-9F antibody may therefore provide a useful marker to distinguish the different lung carcinomas and for investigating the different cells of origin of these tumors.

Study of estrogen receptor, progesterone receptor, and the estrogen-regulated Mr 24,000 protein in patients with carcinomas of the endometrium and cervix.

The presence of an estrogen-regulated protein with 24,000 molecular weight has been studied in 47 patients with endometrial carcinomas and in 29 patients with cervical carcinomas in order to correlate its presence with that of estrogen receptors (ERs) and progesterone receptors (PgRs). In the cytosol tumor samples the Mr 24,000 protein was detected by the Western blot technique using a monoclonal antibody (C11), while the presence of ER and PgR was studied by the one-point dextran-coated charcoal assay. In the tumor tissue sections immunohistochemistry was applied to detect Mr 24,000 protein, ER, and PgR; in these cases monoclonal antireceptor antibodies (H222 and mPRI) were used to localize the receptor proteins. In endometrial and endocervical adenocarcinomas the presence of Mr 24,000 protein correlated significantly with that of ER (P less than or equal to 0.05) in the cytosol samples; when the evaluation was performed in the tumor sections, the presence of Mr 24,000 protein correlated with that of ER (P less than or equal to 0.005) and PgR (P less than or equal to 0.05) as well. The study also showed that almost 70% of the well-differentiated adenocarcinomas had ER, PgR, and Mr 24,000 protein. In 25% of the endometrial adenocarcinomas examined the tumors were associated with normal, proliferative, and hyperplastic endometrium; in these cases the presence of ER, PgR, and Mr 24,000 protein was evaluated by immunohistochemistry in the malignant and nonmalignant endometrium. On the other hand, there was a lack of correlation between Mr 24,000 protein, ER, and PgR in the squamous carcinomas of the uterine cervix and in the endometrial adenocarcinomas with squamous cells. In most of these cases the tumors lacked ER and PgR although 80% of them contained the Mr 24,000 protein to a variable degree. It is suggested that Mr 24,000 protein is involved in growth and differentiation (the Mr 24,000 protein is a heat shock protein) and that the gene coding of this protein is under hormonal control only in those tissues where growth and differentiation are strongly hormonally controlled (breast and endometrium).

Establishment and characterization of two new squamous cell carcinoma cell lines derived from tumors of the head and neck.

Two human cell lines were established from untreated squamous cell carcinomas of the head and neck. Line 183 was derived from a head and neck squamous cell carcinoma of the tonsil and 1483 from a head and neck squamous cell carcinoma of the retromolar trigone. Both lines grow in a cobblestone pattern demonstrating their epithelial heritage. Immunofluorescence studies and one-dimensional polyacrylamide gel electrophoresis indicated that both lines contain cytokeratins. Line 1483 is more aggressive in nude mice, has a higher efficiency for anchorage-independent growth, expresses p21ras (product of the ras oncogene) at a higher level, and is more aneuploid than 183. 1483 also grows as a multicellular tumor spheroid. Line 1483, which was established from the primary tumor of a patient with nodal metastasis, thus displays more progressed characteristics than line 183, which was established from a patient with no clinically positive nodes.

Responses of bone marrow gamma-glutamyltranspeptidase and alkaline phosphatase in vitro to tumor-elaborated granulocytopoietic factors.

Evidence has been obtained for the humoral mediation of the recently noted tumor-induced rise of the host bone marrow gamma-glutamyltranspeptidase (gamma GT) and alkaline phosphatase (AP) content in vivo: normal rat bone marrow suspensions, if incubated for 18 hr to 3 days with serum from mammary carcinoma hosts, show 2- to 8-fold elevations (per cell) of the same 2 enzymes. The active substance(s) is in the acid-stable, HCI-ethanol-soluble polypeptide fraction of the mammary carcinoma extract, and of the hosts' blood serum. The larger the size of the neoplasm, and the faster its growth rate, the greater the effect of the host serum on the gamma GT and AP of the normal bone marrow cells. In host rats in vivo, this response is followed by increases in the number (as well as the gamma GT and AP content) of circulating granulocytes. Therefore, a positive response on the part of these enzymes in the bone marrow suspension was also sought, and found, upon incubation with preparations which enhance granulocyte colony formation in agar cultures (i.e., colony-stimulating factor and serum from endotoxin-treated rats). The results indicate: (a) that the increase in gamma GT and AP is a necessary prelude to stimulation of granulocyte multiplication by appropriate growth factors; and (b) that measurement of these enzymes in the short-term liquid culture offers a biochemical test for such factors elaborated by cancers or in nonneoplastic conditions.