Synthesis and characterization of multiply-tyrosinated, multiply-iodinated somatostatin analogs.

Radio-labeled somatostatin analogs have recently gained popularity as agents useful in intraoperative tumor localization, external scintigraphy and in situ radiotherapy. We have synthesized and characterized a series of novel N-terminally extended multiply-tyrosinated somatostatin analogs that possess high binding affinity for somatostatin receptors, exhibit biological activity comparable to the native peptide and retain these characteristics after iodination. These analogs can be radio-iodinated to high specific activities. Following radioiodination, these analogs exhibit minimal radiolysis and may be clinically useful for tumor localization, scanning and therapy.

Expression of a New Cellular Protein by Monocytoid B-Lymphocytes Differentiated from the Acute Lymphoblastic Leukemia Cell Line (REH).

In an attempt to identify differentiation-related changes in cellular proteins, the common acute lymphoblastic leukemia (ALL) cell line, REH, was studied. REH cells were cultured in either the absence or presence of 12-0-tetradecanoylphorbol-13-acetate (TPA). Changes in surface phenotype were analyzed using monoclonal antibodies and flow cytometry. Cellular proteins were analyzed with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of [(3)H]leucine and [(32)P]orthophosphate labeled cells. Immunophenotype and 2D-PAGE studies were replicated three times on untreated (control) and TPA-treated cells on day 5. Immunophenotypic analysis showed that TPA induced further differentiation of REH cells along the B-cell lineage as indicated by significant decrease in the expression of CD10, induction of CD11c and increase in the expression of CD22. 2D-PAGE of [(3)H]leucine but not the [(32)P]orthophosphate showed that TPA induced the expression of a unique protein. The apparent relative molecular mass (Mr) of the resolved protein was ~23 kd with a ~6.2 isoelectric point (PI). Based on the morphologic and phenotypic findings, our data suggest that the new protein (p23-6.2) and the quantitative changes in protein synthesis induced by TPA are differentiation-related. Our study also indicates that 2D-PAGE analysis is a sensitive and complementary tool to phenotypic markers in the study of differentiation of malignant B-cells.

Isolation and characterization of a spontaneously immortalized human breast epithelial cell line, MCF-10.

Two sublines of a breast epithelial cell culture, MCF-10, derived from human fibrocystic mammary tissue exhibit immortality after extended cultivation in low calcium concentrations (0.03-0.06 mM) and floating transfers in low calcium (MCF-10F), or by trypsin-Versene passages in the customary (normal) calcium levels, 1.05 mM (MCF-10A). Both sublines have been maintained as separate entities after 2.3 years (849 days) in vitro and at present have been in culture for longer than 4 years. MCF-10 has the characteristics of normal breast epithelium by the following criteria: (a) lack of tumorigenicity in nude mice; (b) three-dimensional growth in collagen; (c) growth in culture that is controlled by hormones and growth factors; (d) lack of anchorage-independent growth; and (e) dome formation in confluent cultures. Cytogenetic analysis prior to immortalization showed normal diploid cells; although later passages showed minimal rearrangement and near-diploidy, the immortal cells were not karyotypically normal. The emergence of an immortal culture in normal calcium media was not an inherent characteristic of the original tissue from which MCF-10 was derived since reactivated cryo-preserved cells from cultures grown for 0.3 and 1.2 years in low calcium were incapable of sustained growth in normal calcium.

Polypeptide composition of normal and neoplastic human breast tissues and cells analyzed by two-dimensional gel electrophoresis.

The protein populations of epithelial cells cultured from two neoplastic and five non-neoplastic human breast tissues were resolved and displayed by two-dimensional polyacrylamide gel electrophoresis and silver-staining. With a computer-based image analysis system, we identified eight polypeptides which are present in both of the neoplastic cell lines, but absent from all five of the cultures of non-neoplastic breast cells. The eight polypeptides are not unique to cells cultured from neoplastic breast, because they are also found in cells cultured from non-breast tissues, both neoplastic and non-neoplastic. Two of the eight polypeptides (approximately Mr 25,000/pI 4.4 and approximately Mr 31,000/pI 5.5) are present in the patterns of whole tissue samples from infiltrating ductal carcinomas and absent in most normal breast tissue.

Evidence for a direct growth-stimulating effect of estradiol on human MCF-7 cells in vivo.

The MCF-7 continuous line of human breast cancer cells requires that athymic nude mice receive supplemental estrogen so that inocula can produce progressively growing tumors. Although these cells contain a typical estrogen receptor complex, the lack of consistent growth stimulation induced by estrogens added to in vitro culture systems has raised the question as to whether this class of hormones acts directly upon the cells or induces a second message produced in other tissues. The present experiments were designed to test the effect of estradiol on the growth of these cells in vivo by exposing them directly to the hormone prior to its absorption into the hepatic portal circulation and subsequent metabolic inactivation. Tumor fragments that were placed next to an estradiol-containing pellet in the spleen grew to produce grossly evident tumor masses, whereas those in the subcutis of the same animals did not, although some minute residua did remain. In the splenic tumors, the mitotic index of the MCF-7 cells immediately adjacent to the estrogen pellets was 2.4 times that of cells on the other side of the same tumor and 3.5 times that of those in the minute s.c. residua. We interpret these data as indicating that in vivo estradiol is acting directly upon the MCF-7 cells to increase their rate of proliferation rather than to initiate the production of a second message to be released into the circulation. Additionally, it was found that s.c. tumors that were decreasing in volume subsequent to withdrawal of systemic estrogen still contained dividing neoplastic cells but with a lower frequency than that seen in progressively growting tumors stimulated with estradiol. This finding indicates that MCF-7 cells can proliferate in vivo in the absence of a substantial amount of estrogen but only at a rate insufficient to sustain progressive tumor growth.

Induction of endogenous mammary tumor virus expression during hormonal induction of mammary adenoacanthomas and carcinomas of BALB/c female mice.

Treatment of BALB/c female mice with pituitary isografts, under conditions that established mammary hyperplasia in an anovulatory condition, enhanced mammary tumor development with prior onset of dysplastic foci in lobular parenchyma. Tumor onset began at 8 months (mean onset time, 18 mo); the 25-month incidence was 100%. Adenoacanthomatous tumors appeared first. Adenocarcinomas appeared only after more than 14 months of continuous hormone stimulation. Dysplastic and neoplastic changes occurred while blood levels of the three major mammotropic hormones were physiologic. Murine mammary tumor virus (MuMTV) p28 was detected in all tumors tested, independent of time of tumor appearance or tumor type, although keratinizing cells in adenoacanthomatous tumors did not contribute to MuMTV antigen expression. MuMTV gp52 was detected in only a small fraction of cells in a few tumors. MuMTV RNA contents were above normal in all tumors tested. Neither MuMTV structural antigens nor RNA was induced in normal glands by the same hormone treatment that ultimately resulted in dysplasia and tumor formation and elevated levels of these viral markers in neoplasms. The MuMTV RNA in all hormone-induced tumors was readily distinguishable in base sequence from standard MuMTV RNA but indistinguishable from MuMTV RNA recovered from lactating mammary glands of BALB/c females carrying only endogenous MuMTV proviruses, suggesting that endogenous MuMTV RNA sequences were induced in hormone-induced neoplasms. RNA indistinguishable from MuMTV sequences present in hormone-induced primary tumors was also detected in multiple genomic equivalents in two independently derived hormone-induced premalignant alveolar hyperplasias. MuMTV p28 was detected, but gp52 was not. The same hormone stimulus that generated 100% tumors in normal gland greatly accelerated tumor development in premalignant hyperplasias but did not amplify MuMTV RNA or antigens in either hyperplasias or the tumors derived from them. B-type virions were not detected in these tissues, in either hypophyseal implant-stimulated or virgin hosts. Cell-free virions were not detected in culture. These data suggest that the replication of MuMTV induced in hormone-induced neoplasms is defective. Details of its expression suggest that if involved in events leading to tumors, its involvement is insufficient cause for those tumors.

Effects of using long breathing hoses upon mask pressure.

Effects of using oxygen breathing hoses from 0.9 to 8.2 m (3 to 27 ft) long and mask fit upon mask pressure during 0.75 to 12-s decompressions from 2,438 m (8,000 ft) to either 6,096, 10,668, or 15,240 m (20,000, 35,000 or 50,000 ft) were determined. Peak mask pressures and duration of high mask pressure were related to mask fit, mask and hose stretch compliance, pressure differential, decompression rate, and other factors, with mask pressure increasing with hose length. Peak mask pressures frequently exceeded 80 mm Hg, a high pressure associated with increased incidence of pulmonary damage. Cargo-type aircraft, however, have sufficiently large volumes so that they will not decompress rapidly enough to have high mask pressure, even with an 8.2-m long hose. Long breathing hoses should not be used in smaller aircraft since small cabin volume will result in rapid decompression rates and high mask pressure. Above a flight altitude of 2,438 m, oxygen should always be breathed if hoses longer than 2.9 m (9 ft) are used. This would help prevent hypoxia, associated with the need to deplete air in the hose before oxygen is breathed, should cabin pressure be lost at a high altitude. The fastest decompression rates compatible with preventing mask pressures from exceding 80 mm Hg during decompressions to different altitudes with different length breathing hoses are given.