Atmospheric CO2 levels from 2.7 billion years ago inferred from micrometeorite oxidation.

Earth's atmospheric composition during the Archean eon of 4 to 2.5 billion years ago has few constraints. However, the geochemistry of recently discovered iron-rich micrometeorites from 2.7 billion-year-old limestones could serve as a proxy for ancient gas concentrations. When micrometeorites entered the atmosphere, they melted and preserved a record of atmospheric interaction. We model the motion, evaporation, and kinetic oxidation by CO2 of micrometeorites entering a CO2-rich atmosphere. We consider a CO2-rich rather than an O2-rich atmosphere, as considered previously, because this better represents likely atmospheric conditions in the anoxic Archean. Our model reproduces the observed oxidation state of micrometeorites at 2.7 Ga for an estimated atmospheric CO2 concentration of >70% by volume. Even if the early atmosphere was thinner than today, the elevated CO2 level indicated by our model result would help resolve how the Late Archean Earth remained warm when the young Sun was ~20% fainter.

The antiquity of oxygenic photosynthesis: evidence from stromatolites in sulphate-deficient Archaean lakes.

The Tumbiana Formation, about 2700 million years old, was largely deposited in ephemeral saline lakes, as judged by the unusual evaporite paragenesis of carbonate and halite with no sulfate. Stromatolites of diverse morphology occur in the lacustrine sediments, some with palimpsest fabrics after erect filaments. These stromatolites were probably accreted by phototropic microbes that, from their habitat in shallow isolated basins with negligible sulfate concentrations, almost certainly metabolized by ozygenic photosynthesis.

Archean molecular fossils and the early rise of eukaryotes.

Molecular fossils of biological lipids are preserved in 2700-million-year-old shales from the Pilbara Craton, Australia. Sequential extraction of adjacent samples shows that these hydrocarbon biomarkers are indigenous and syngenetic to the Archean shales, greatly extending the known geological range of such molecules. The presence of abundant 2alpha-methylhopanes, which are characteristic of cyanobacteria, indicates that oxygenic photosynthesis evolved well before the atmosphere became oxidizing. The presence of steranes, particularly cholestane and its 28- to 30-carbon analogs, provides persuasive evidence for the existence of eukaryotes 500 million to 1 billion years before the extant fossil record indicates that the lineage arose.

Isolation of a cDNA corresponding to a developmentally regulated transcript in rat intestine.

We report the isolation of a cDNA clone corresponding to a transcript that is accumulated differentially in rat intestine during development. Clone OCI-5 was selected from the rat intestinal cell line IEC-18, which represents primitive intestinal epithelial crypt cells. Expression was high in rat fetal intestine between 15 and 19 days of development and thereafter was progressively down regulated, becoming undetectable after weaning. Clone OCI-5 detected homologous sequences in human and murine cells. In particular, a high level of expression was detected in CaCo-2, a human colon carcinoma cell line, which is known to express molecules characteristic of fetal small intestinal cells. Expression of a homologous gene was also detected in F9 murine teratocarcinoma cells when they were induced to differentiate into parietal or visceral endodermlike cells. When IEC-18 cells were transformed by activated H-ras or v-src genes, expression of clone OCI-5 was suppressed; the degree of down-regulation correlated with the extent of morphological change induced in the transformed IEC-18 cells. The sequence of clone OCI-5 showed an open reading frame that was capable of encoding a protein of 597 amino acids, but no strong homology was found with any of the proteins registered in the protein sequence data base.

Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar.

Environmental niches and metabolic diversity in Neoarchean lakes.

The diversification of macro-organisms over the last 500 million years often coincided with the development of new environmental niches. Microbial diversification over the last 4 billion years likely followed similar patterns. However, linkages between environmental settings and microbial ecology have so far not been described from the ancient rock record. In this study, we investigated carbon, nitrogen, and molybdenum isotopes, and iron speciation in five non-marine stratigraphic units of the Neoarchean Fortescue Group, Western Australia, that are similar in age (2.78-2.72 Ga) but differ in their hydro-geologic setting. Our data suggest that the felsic-dominated and hydrologically open lakes of the Bellary and Hardey formations were probably dominated by methanogenesis (δ13 Corg  = -38.7 ± 4.2‰) and biologic N2 fixation (δ15 Nbulk  =-0.6 ± 1.0‰), whereas the Mt. Roe, Tumbiana and Kylena Formations, with more mafic siliciclastic sediments, preserve evidence of methanotrophy (δ13 Corg as low as -57.4‰, δ13 Ccarb as low as -9.2‰) and NH3 loss under alkaline conditions. Evidence of oxygenic photosynthesis is recorded only in the closed evaporitic Tumbiana lakes marked by abundant stromatolites, limited evidence of Fe and S cycling, fractionated Mo isotopes (δ98/95 Mo = +0.4 ± 0.4‰), and the widest range in δ13 Corg (-57‰ to -15‰), suggesting oxidative processes and multiple carbon fixation pathways. Methanotrophy in the three mafic settings was probably coupled to a combination of oxidants, including O2 and SO42- . Overall, our results may indicate that early microbial evolution on the Precambrian Earth was in part influenced by geological parameters. We speculate that expanding habitats, such as those linked to continental growth, may have been an important factor in the evolution of life.

A stem cell model of human tumor growth: implications for tumor cell clonogenic assays.

A simple stem cell model of human tumor growth is presented. Three tumor cell populations are predicted: stem, transitional, and end cells. The properties of these cells are discussed in terms of their behavior in currently available technologies for investigation of cell kinetics and for their influence on clinical outcome. Stem cell renewal, transitional cell proliferation, and cell loss are analyzed mathematically to define their influence on the relative proportions of cell populations; it is demonstrated that stem cell renewal has a central role in determining the growth properties of tumors. The impact of a stem cell model on the use of tumor clonogenic assays as predictors of clinical outcome is discussed; opinions are expressed as to the definition of reasonable expectations for current experimental procedures.

Cytotoxicity of adriamycin and daunorubicin for normal and leukemia progenitor cells of man.

A colony assay available for a subpopulation of acute myeloblastic leukemia blasts with proliferative potential was used to measure adriamycin (adria) and daunorubicin (dauno) dose-response curves following brief exposure to either drug and washing. The dose-response curves were simple negative exponentials that might be characterized by D10 (dose required to reduce survival to 10%) values. The D10 values ranged from 0.47 to 20.8 microgram/ml for adria (8 patients) and from 0.06 to 0.34 microgram/ml for dauno (3 patients). Controls consisted of committed granulopoietic and T-lymphocyte progenitors. Four measurements of granulopoietic progenitors yielded D10 values from 2.5 to 11.5 mug/ml for adria and from 0.44 to 1.2 microgram/ml for dauno. T-lymphocyte precursors from 4 normal individuals were resistant. However, following incubation of normal leukocytes with phytohemagglutinin, DNA synthesis commenced in T-lymphocyte precursors for 3 additional normal controls, which was associated with an increased data sensitivity with D10 values ranging from 4.4 to 6.2 microgram/ml.

Regulation of transforming growth factor beta receptors in H-ras oncogene-transformed rat intestinal epithelial cells.

We have reported that over expression of the H-ras oncogene causes resistance to growth inhibition by transforming growth factor beta 1 (TGF-beta 1) and a time-dependant switch of type II to type I TGF-beta receptor expression in the rat intestinal epithelial cell line IEC-18 (J. Filmus, J. Zhao, and R. N. Buick, Oncogene, 7: 521-526, 1992). Here, we investigate the possible mechanisms involved in H-ras-mediated regulation of TGF-beta receptors in an IEC-18 cell clone expressing H-ras, conditional on the activity of a dexamethasone-sensitive promoter. The switch from type II to type I receptor expression in response to H-ras expression has a requirement for de novo RNA synthesis. In addition, accumulation of TGF-beta receptor type II mRNA is approximately 5-fold lower in ras-expressing cells compared to control cells. Nuclear run-on experiments suggest that the down-regulation of type II receptor mRNA by H-ras oncogene is based, at least in part, on reduced transcription. We have also analyzed the consequences of H-ras expression on the properties of the TGF-beta receptors. Type I and II in IEC-18 cells and type I receptors in ras-transformed cells have similar characteristics in terms of binding affinities for TGF-beta 1 (or TGF-beta 2) turnover rates and glycosylation states. Notably, the type I receptors in ras-transformed cells are not capable of ligand-induced internalization. Although H-ras expression in IEC-18 cells causes resistance to TGF-beta-mediated growth inhibition, the cells remain responsive to TGF-beta 1 stimulation of fibronectin expression. These results are discussed in the context of the knowledge of TGF-beta receptor complexity and signal transduction, and with reference to the potential role for loss of TGF-beta-mediated negative growth regulation in malignant transformation.

Relationship of c-myc expression to differentiation and proliferation of HL-60 cells.

We have compared changes in c-myc expression in HL-60 promyelocytic leukemia cells induced to differentiate by dimethyl sulfoxide or growth inhibited in an undifferentiated state. Under these conditions, c-myc expression did not correlate with the proportion of proliferating cells. The kinetics of the decrease in c-myc expression upon differentiation induction is paralleled closely by an increasing proportion of histochemically detected differentiated myeloid cells and by a decrease in clonogenic potential but not by changes in the proportion of proliferating cells. Changes in c-myc expression subsequent to differentiation induction can therefore be directly related to the differentiation process rather than to a cell cycle-related phenomenon.

Stability of c-K-ras amplification during progression in a patient with adenocarcinoma of the ovary.

We have identified a case of serous cystadenocarcinoma of the ovary in which the tumor cells display an amplification (from 10- to 20-fold) of the cellular oncogene K-ras. Normal cells purified from the malignant ascites did not show such amplification. Five consecutive samples were obtained by paracentesis over a 9-month period during which the patient received chemotherapy and underwent clinical progression. The level of c-K-ras amplification in the tumor cells did not change during this period. In studies of the tumors of 6 additional patients with adenocarcinoma of the ovary and 5 cell lines of the same histology, we have detected no other example of significant c-K-ras amplification.

Preparation of permanent slides of intact soft-agar colony cultures of hematopoietic and tumor stem cells.

A simple technique is described for fixing colony-containing layers of soft agar and drying them onto microscopic slides. The method is extrapolated from techniques used in immunology for permanent preservation of immunodiffusion or immunoelectrophoresis plates. Slides prepared in this fashion are eminently suitable for subsequent analysis with a variety of techniques including conventional Papanicolaou or other staining methods as well as histochemistry, immunofluorescence, and autoradiography. In addition to research applications, the technique may have diagnostic applications and should greatly enhance both qualitative and quantitative analysis of the biology of hematopoietic and tumor colony formation.

Cellular specificity of NB70K, a putative human ovarian tumor antigen.

We have studied the cellular expression of NB/70K, a glycoprotein proposed to be human ovarian tumor associated described previously. Analysis of cells bearing this antigen in malignant ascites shows expression in tumor cells of gynecological origin (ovarian, endometrial) and to a limited degree in breast cancer cells. Within such tumors, there is a weak inverse correlation between labeling index and antigen expression. Furthermore, evidence is presented to show that the cells bearing this glycoprotein are physically separable from those bearing carcinoembryonic antigen.

Comparative properties of five human ovarian adenocarcinoma cell lines.

We describe the derivation of three human ovarian carcinoma cell lines and the comparison of their properties with two previously described cell lines of like histology (SKOV-3 and CAOV-3). Two of the new lines (HOC-1 and HOC-7) were derived from separate ascites tumors (at 9-month intervals) of a patient with well-differentiated serous adenocarcinoma of the ovary. The third new line, HEY, was derived from a human ovarian cancer xenograft (HX-62) originally grown from a peritoneal deposit of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary. The cell lines demonstrated differential ability to grow in semisolid culture and as xenografts in immunologically deprived CBA/CJ mice. Dose-response curves were generated for clonogenic cell survival of cells exposed to common chemotherapeutic agents; one of the lines (HEY) shows a degree of resistance to the alkylating agent cis-diamminedichloroplatinum(II) (cis-platinum). Common karyological features included structural abnormalities of chromosomes 3 and 11. Heterogeneity of expression of ovarian tumor-associated antigens was documented.

A cell line from a human ovarian carcinoma with amplification of the K-ras gene.

We have recently reported that ascitic cells from a human ovarian carcinoma have a 10- to 20-fold K-ras amplification and that the level of such amplification did not change over a 9-month period during which the patient received chemotherapy and underwent clinical progression. Here we describe an ovarian tumor cell line (HOC-8) which has been derived from that tumor and which also shows a similar level of K-ras amplification. The amounts of K-ras specific mRNA and the Mr 21,000 protein encoded by the amplified gene are correspondingly elevated. karyotypic analysis revealed no detectable double minute chromosomes but did show an abnormal banding region on chromosome 6. This cell line may represent a useful model to investigate the significance of the K-ras gene product for the pathogenesis of human tumors.

Tumor progression studied by analysis of cellular features of serial ascitic ovarian carcinoma tumors.

Seven consecutive ascitic tumors were obtained over a 9-month period from a patient with serous adenocarcinoma of the ovary. The tumor cell populations were analyzed for cellular proliferation (labeling index, agar clonogenicity, and self-renewal capacity), for cell differentiation (cell surface expression of carcinoembryonic antigen and histochemical stain for fat accumulation), and for karyotypic changes. Evidence is presented of increased aggressiveness of proliferative features together with a decreasing proportion of cells with differentiated features. Parallel temporal changes were documented in density-volume characteristics of the tumor cell population, from small, high-density to large, low-density cells. The only karyotypic change identified over this period was the loss of one X-chromosome and the increased frequency of cells containing double minute bodies. The progressive characteristics described in this human tumor are not, therefore, associated with gross chromosomal changes. The accumulation of double minute chromosome bodies may be associated with a low-dose methotrexate exposure or with the tumor progression.

Self-renewal capacity of leukemic blast progenitor cells.

A review is presented of experimental information pertaining to the characteristics of a procedure designed to quantitate the capacity for self-renewal in clonogenic cells of human acute myeloblastic leukemia. In a series of 44 previously untreated patients, a significant correlation (p less than 0.01) was seen between low capacity for self-renewal and successful remission induction. Three cytotoxic drugs (Adriamycin, 1-beta-D-arabinofuranosylcytosine, and N-[4-(19-acridinylamino)-3-methoxyphenyl]-methanesulfonamide) were tested for preferential effect against self-renewal events. Surviving clonogenic cells to these agents had, respectively, unchanged, lower, and higher capacity for self-renewal. The implications of such drug properties are discussed.

Development of an agar-methyl cellulose clonogenic assay for cells in transitional cell carcinoma of the human bladder.

We report the development of a clonogenic assay for progenitor cells in transitional cell carcinoma of the bladder. Colony growth has been demonstrated from cells obtained both from surgical biopsies and from bladder barbotages. Electron microscopic and karyotypic evidence supports the contention that these progenitors represent a part of the population maintaining the tumor in vivo. Colony growth occurred in 9 of 11 surgical biopsy samples and in 6 of 6 bladder barbotage samples. Plating efficiency ranged up to 0.7%, and colony size was in some instances greater than 1000 cells. The assay appears potentially useful for analysis of the biology of human transitional cell carcinoma.

Monoclonal antibodies to an epithelial ovarian adenocarcinoma: distinctive reactivity with xenografts of the original tumor and a cultured cell line.

Four monoclonal antibodies (mAb) (8C, 10B, M2A, and M2D) were produced against the human epithelial ovarian adenocarcinoma cell line, HEY. The affinity constants of binding of the mAb to cultured HEY cells were 8 X 10(8) M-1 (M2D) and 10(9) M-1 (8C and 10B). mAb 8C reacted with a major glycoprotein of Mr 90,000 on the surface of HEY cells. The four mAb differed from previously reported mAb to epithelial ovarian adenocarcinomas on the basis of their reactivity with cultured ovarian adenocarcinoma cell lines using a cell-binding radioimmunoassay, and their staining of cryostat sections of various human normal and tumor tissues using an immunoperoxidase reaction. All four mAb reacted with s.c. tumors derived by injecting cultured HEY cells into thymectomized CBA/CJ mice. However, only two of the four mAb (8C and 10B) also reacted with s.c. tumors of the original HEY xenograft from which the cultured cell line was derived. In addition, mAb 8C and 10B reacted by immunoperoxidase staining with 2 and 4 different cases, respectively, of 11 epithelial ovarian adenocarcinomas examined. Cultured HEY cells were adapted to grow i.p. in BALB/c-nu/nu mice and the i.p. tumors retained their reactivity with the monoclonal antibodies. These tumor-bearing mice offer a useful model system for studying the potential of mAb, especially 8C and 10B, for the diagnosis and treatment of patients with peritoneal extension of epithelial ovarian adenocarcinomas.

Varying degrees of amplification of the N-ras oncogene in the human breast cancer cell line MCF-7.

The oncogene N-ras has been found to be amplified (congruent to 20 copies) in the human breast carcinoma cell line MCF-7. The amplified sequences have been localized to a marker chromosome by in situ hybridization. Sublines of MCF-7, serially passaged in different laboratories, have marked variation in the degree of N-ras amplification. The differing degrees of amplification of N-ras are further evidence of heterogeneity within MCF-7 subclones. The phenomenon may not have general relevance for breast cancer, since other breast cancer cell lines and DNA from patient biopsies failed to show evidence of N-ras amplification.