Metformin inhibits breast cancer cell growth,colony formation and induces cell cycle arrest in vitro

The anti-diabetic drug metformin reduces human cancer incidence and improves the survival of cancer patients, including those with breast cancer. We studied the activity of metformin against diverse molecular subtypes of breast cancer cell lines in vitro. Metformin showed biological activity against all estrogen receptor (ER) positive and negative, erbB2 normal and abnormal breast cancer cell lines tested. It inhibited cellular proliferation, reduced colony formation and caused partial cell cycle arrest at the G1 checkpoint. Metformin did not induce apoptosis (as measured by DNA fragmentation and PARP cleavage) in luminal A, B or erbB2 subtype breast cancer cell lines. At the molecular level, metformin treatment was associated with a reduction of cyclin D1 and E2F1 expression with no changes in p27kip1 or p21waf1. It inhibited mitogen activated protein kinase (MAPK) and Akt activity, as well as the mammalian target of rapamycin (mTOR) in both ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells. In erbB2-overexpressing breast cancer cell lines, metformin reduced erbB2 expression at higher concentrations, and at lower concentrations within the therapeutic range, it inhibited erbB2 tyrosine kinase activity evidenced by a reduction of phosphorylated erbB2 (P-erbB2) at both auto- and Src- phosphorylation sites. These data suggest that metformin may have potential therapeutic utility against ER positive and negative, erbB2-overexpressing and erbB2-normal expressing breast cancer cells.     

Low density lipoprotein receptor binding in aging human diploid fibroblasts in culture

High affinity cell surface receptors for low density lipoproteins (LDL) are inducible in cultured human lung fibroblasts by the removal of lipoproteins from the cell culture medium. The binding, uptake, and degradation of 125I-LDL by fibroblasts decrease with increasing number of population doublings. The affinity of LDL receptor binding, however, remained unchanged at different population doublings levels. Hence, the difference in LDL binding activity in the aging fibroblasts can be attributed to a reduction in the number of receptor sites on the cell membrane. Cellular uptake of [4-14C]cholesterol and 2-deoxy-D-[1-14C]glucose mediated through mechanisms independent of the LDL receptor pathway revealed no significant difference in early and late passage fibroblasts. This suggests that the alteration in the LDL receptor binding in serially passaged fibroblasts is an "age-related" phenomenon. The late population doubling fibroblasts require more LDL in the culture medium for feedback inhibition of LDL receptor synthesis. Thus, aging fibroblasts are both progressively less inducible and less suppressible in the regulation of their cell membrane LDL receptors. Similar results were also obtained with respect to the regulation of DL-3-hydroxy-3-methyl-glutaryl coenzyme A reductase in the aging fibroblasts in culture; the enzyme has become less inducible and less supressible as the fibroblasts approach the limit of their in vitro lifespan. These age-related alterations in the cellular metabolism of LDL and cholesterol might contribute to our understanding of the increased risk of athlerosclerosis in our aging population.     

Comparison of features of carcinogen-transformed human cells in vitro with sarcoma-derived cells

To define characteristics of chemically transformed phenotypes during and after progression to neoplasia and to assess their relationship to those phenotypes expressed by surgically removed sarcoma lesions, we compared the characteristics in the following manner. We investigated: (1) alterations in growth patterns; (2) anchorage-independent growth; (3) reactivity with monoclonal antibodies directed against surface antigen; (4) invasiveness in embryonic chick skin; (5) tumorigenicity in nude mice; and (6) karyology. Fifty different sarcoma cell lines were examined which exhibited different rates and absolute numbers of population doublings. With one exception, all sarcoma cell lines exhibited a finite life span ranging from 60 to 100 population doublings. Populations of these cells that exhibited anchorage-independent growth in soft agar also reacted positively with a monoclonal antibody (MoAb) 345.134S directed against a 115K-GP cell surface glycoprotein. Similarly, chemically transformed cells that grew in soft agar also reacted with the MoAb 345.134S, whereas cells with an inability to grow in soft agar did not. Cell lines established from human sarcoma and from chemically transformed human fibroblasts that reacted positively with the MoAb 345.134S were invasive for embryonic chick skin and formed tumors in nude mice. The selection medium used during culture of the carcinogen-treated cells resulted in the appearance of an altered phenotype that after at least 16 population doublings exhibited characteristics common to those cells derived from human sarcomas.     

In vitro induction and expression of interleukin 2 receptor in a clonal T helper cell differentiation model

Induction and expression of interleukin 2 (IL 2) receptor have been studied using a poly( Glu60 Ala30 Tyr10 ) (GAT)-specific T cell clone of mouse origin. This clone (52-3) has been characterized and it exhibits functional properties of T helper (TH) cells: it leads to a specific anti-DNP response in the presence of DNP-GAT and DNP-primed B cells and it secretes biological activities which can induce polyclonal B cell proliferation and IgM secretion. In vitro this clone mimics the activation stages of normal T lymphocytes and can be obtained under two states of differentiation. depending on the antigen-specific signal provided by antigen-presenting cells (APC). The expression of IL 2 receptor by this clone has been studied by (i) its response to IL 2, (ii) its capacity to absorb IL 2 bioactivity, and (iii) its reactivity with monoclonal antibody 7D4 specific for mouse IL 2 receptor. All the results indicate that the unstimulated state does not express the IL 2 receptor while the activated state does. Clone 52-3 has been compared with clone 14-1.6 that derives from a TH cell line and expresses the IL 2 receptor constitutively. 52-3 offers a good experimental model for studying in vitro, in a clonal TH cell population, the detailed mechanism of IL 2 receptor induction.     

Effects of density and cellular aging on collagen synthesis and growth kinetics in keloid and normal skin fibroblasts

Summary Cell proliferation and collagen synthesis were analyzed in high, medium, and low density keloid and normal skin fibroblasts and also at various times during the course of in vitro aging to expand previous findings of increased collagen synthesis in keloid compared to normal skin fibroblasts. It was found that both keloid and normal fibroblasts (<20 population doublings) responded similarly to high, medium, and low initial plating densities; however, sparsely plated keloid fibroblasts exhibited a loss of replicative capacity earlier in their in vitro lifespan than did sparsely plated normal skin fibroblasts. When analyzed at population doubling levels 2 to 38, collagen synthesis was elevated in keloid compared to normal skin fibroblasts but decreased at the same rate in both cell types throughout this in vitro interval. Supported by NIH Grant GM-20298.     

Influence of tissue origins and external microenvironment on porcine foetal fibroblast growth, proliferative life span and genome stability

One of the challenges of manipulating genes in primary cells is that the cells have a finite proliferation capacity. This, combined with the lower gene targeting efficiency of somatic cells, makes identification of targeted clones very difficult. The objective of this study was to establish a system that allows porcine foetal fibroblasts to reach their maximal proliferation capacity in vitro. The influence of fibroblast origin, stage of foetal development, cell seeding densities and concentration of foetal bovine serum (FBS) on the population doublings, the percentage of beta-galactosidase-activity-positive cells and the genome stability of foetal fibroblasts during in vitro culture was investigated. It was found that porcine foetal fibroblasts could be cultured for over 80 population doublings in the appropriate culture system. Fibroblasts from earlier stages of foetal development were better candidate cells than those from the later stages. Cells from the heart were more actively proliferative and more resistant to replicative senescence than those from the liver. Compared to 10% FBS content, 15% FBS provided better homeostatic support, not only to proliferative performance, but also in maintaining a normal karyotype. In addition, the proliferative life span of porcine foetal fibroblasts is also dependent on seeding density of the culture.     

The time-pattern of rises and falls in proliferation fades with senescence of mortal lines and is perpetuated in immortal rat hepatoma fao cell line

Summary Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3–4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22–23 additional population doublings each. Normal embryonic fibroblasts culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attribution and repair processes regulates the waves of proliferation. Equal contributors to these studies.     

The effect of choleragen and epidermal growth factor on proliferation and maturation in vitro of human ectocervical cells

Summary The role of choleragen (CT) and epidermal growth factor (EGF) has been examined in relation to the control of growth and differentiation of adult human cervical epithelial (HCE) cells derived from the ectocervix. Cervical biopsies derived from hysterectomy specimens were trypsin disaggregated and HCE cells were plated at 5×10³/cm² in the presence of 2×10⁴/cm² lethally irradiated Swiss 3T3 fibroblasts. Cultures were grown in Liebovitz medium supplemented with 10% fetal bovine serum and hydrocortisone. Epidermal growth factor at 10 ng/ml and choleragen at 10⁻¹⁰ M were added to cultures either singly or in combination. DNA replication in these cultures was measured autoradiographically after exposing cells to tritiated thymidine for 2 h. Differentiation was assessed histochemically by determining glycogen accumulation using the periodic acid Schiff technique. Choleragen increased colony plating efficiency by at least a factor of two but had no effect on colony size Epidermal growth factor did not increase plating efficiency but did increase colony size. In EGF treated colonies DNA replication occurred throughout the colony compared to CT treated colonies in which replication was restricted to the periphery. In the absence of EGF, population doublings achieved in culture did not exceed 32 and glycogen accumulation was evident in cells early in culture life. Colonies treated with EGF exhibited glycogen accumulation late in culture life and the EGF treated cells achieved at least 50 population doublings in culture. The results are discussed in relation to the role of EGF and choleragen on cell differentiation.     

Slowed growth of cultured fibroblasts from human radiation wounds

To study radiation effect separate from the microcirculation, fibroblasts were cultured from four patients with radiation wounds. Cells could be grown from irradiated tissue near the ulcer and from control normal tissue, but no cells could be cultured from the ulcers. The ability of radiation-treated fibroblasts to attach to the substrate and form colonies was less than that of unirradiated cells. Irradiated skin fibroblasts from the four patients had significantly longer mean generation times than did control cells. During log-phase growth (1 to 9 days), the population doublings of damaged cells were significantly reduced compared to colonies from normal cells. These data suggest a permanent intrinsic radiation effect on fibroblasts or a selective ablation of faster-growing fibroblast subpopulations that is not dependent on decreased blood supply.     

Effect of fibroblast donor cell age and cell cycle on development of bovine nuclear transfer embryos in vitro

The effects of cell cycle stage and the age of the cell donor animal on in vitro development of bovine nuclear transfer embryos were investigated. Cultures of primary bovine fibroblasts were established from animals of various ages, and the in vitro life span of these cell lines was analyzed. Fibroblasts from both fetuses and calves had similar in vitro life spans of approximately 30 population doublings (PDs) compared with 20 PDs in fibroblasts obtained from adult animals. When fibroblasts from both fetuses and adult animals were cultured as a population, the percentage of cells in G1 increased linearly with time, whereas the percentage of S-phase cells decreased proportionately. Furthermore, the percentage of cells in G1 at a given time was higher in adult fibroblasts than in fetal fibroblasts. To study the individual cells from a population, a shake-off method was developed to isolate cells in G1 stage of the cell cycle and evaluate the cell cycle characteristics of both fetal and adult fibroblasts from either 25% or 100% confluent cultures. Irrespective of the age, the mean cell cycle length in isolated cells was shorter (9.6-15.5 h) than that observed for cells cultured as a population. Likewise, the length of the G1 stage in these isolated cells, as indicated by 5-bromo-deoxyuridine labeling, lasted only about 2-3 h. There were no differences in either the number of cells in blastocysts or the percentage of blastocysts between the embryos reconstructed with G1 cells from 25% or 100% confluent cultures of fetal or adult cell lines. This study suggests that there are substantial differences in cell cycle characteristics in cells derived from animals of different ages or cultured at different levels of confluence. However, these factors had no effect on in vitro development of nuclear transfer embryos.     

Evidence contrary to the protein error hypothesis for in vitro senescence.

A strain of diploid fibroblasts, obtained from the skin of a male infant, was cultured in vitro and cells were tested throughout their lifespan for the appearance of altered glucose-6-phosphate dehydrogenase (G-6-PD) detected either by thermostability studies or by immunotitration. No significant difference was found in the proportion of thermolabile enzyme in 31 young cultures (4.8 +/- 1%, S.E.), in comparison with that in 19 old cultures (4.9 +/- 1%, S.E.). Old cultures had ceased active cell division (49-60 doublings); DNA replication, measured by [3H]thymidine uptake over a period of 24 hours, was limited to less than 5% of these cells. Young cells (5-22 doublings) had a [3H]thymidine labeling index of 75-85%. Titration of G-6-PD activity in extracts of young and old cells with neutralizing antibody directes specifically against G-6-PD failed to detect an increment of enzymatically defective G-6-PD in old cells. The thermostability studies were capable of detecting altered G-6-PD in skin fibroblasts from a female heterozygous for a thermolabile mutant of G-6-PD, and in fibroblasts treated with a proline analogue, azetidine carboxylic acid. The immunotitration technique was also capable of detecting catalytically altered G-6-PD from the thermolabile mutant and G-6-PD inactivated with N-ethylameimide. These findings argue against a protein error catastrophe as the cause of in vitro clonal senescence.     

Establishment and characterization of ferret cells in culture

Cells derived from the brain of a 6 wk-old ferret have been subcultured over 100 times and have undergone over 400 population doublings in vitro. These cells, referred to as Mpf cells, have an absolute efficiency of colony formation in excess of 45%, exhibit a mean population doubling time of 12.5 h, possess ferret-specific antigens, and have isozymes with electrophoretic properties that are the same as those of isozymes found in ferret liver. The cells exhibit a cytopathic effect and support the synthesis of progeny virus when they are infected with the viruses of lymphocytic choriomeningitis, Newcastle disease, pseudorabies, Sindbis, vaccinia, and vesicular stomatitis. The passage level of the Mpf cells, their elapsed number of population doublings, their possession of ferret-specific antigens, and the comigration of four isozymes obtained from these cells and ferret liver define the cells as an established line of ferret cells.     

Establishment and characterization of ferret cells in culture

Summary Cells derived from the brain of a 6 wk-old ferret have been subcultured over 100 times and have undergone over 400 population doublings in vitro. These cells, referred to as Mpf cells, have an absolute efficiency of colony formation in excess of 45%, exhibit a mean population doubling time of 12.5 h, possess ferret-specific antigens, and have isozymes with electrophoretic properties that are the same as those of isozymes found in ferret liver. The cells exhibit a cytopathic effect and support the synthesis of progeny virus when they are infected with the viruses of lymphocytic choriomeningitis, Newcastle disease, pseudorabies, Sindbis, vaccinia, and vesicular stomatitis. The passage level of the Mpf cells, their elapsed number of population doublings, their possession of ferret-specific antigens, and the comigration of four isozymes obtained from these cells and ferret liver define the cells as an established line of ferret cells.     

CHARACTERIZATION OF A BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) KIDNEY EPITHELIAL CELL LINE

Abstract: An epithelial cell line, Carvan dolphin kidney (CDK), isolated from a prematurely born female bottlenose dolphin, Tursiops truncatus , exhibited growth characteristics not previously reported for cetacean cells in culture. CDK cells were cytokeratin positive and demonstrated a maximum doubling time of 1.31 days, with plating and colony forming efficiencies approaching 100% for the early population doublings. Despite an unusually efficient colony-forming ability and rapid growth, these cells were neither transformed nor immortal, displaying normal contact inhibition, anchorage dependence, and the requirement for high concentrations of fetal bovine serum in the growth medium. CDK cells exhibited age-dependent changes in growth rate, colony-forming efficiency, and cytoplasmic profile, and showed a finite lifespan of about 50 population doublings and a stable 2N = 44 karyotype which correlates with previously reported cytogenetic analyses. Velocity sedimentation analysis showed that CDK cells contained nuclear aryl hydrocarbon receptor (Ah), indicating their potential to be induced for cytochrome P450. These data suggest that CDK cells may have utility as an in vitro toxicological model for evaluating hydrocarbon contaminant effects on Tursiops truncatus , a protected marine mammal.     

Aging in vitro : Growth of cultured cells from the Galapagos tortoise

Skin biopsies from two growing and two fully-grown Galapagos tortoises, a species that lives twice as long as man, were explanted in vitro. Cellular outgrowth was more vigorous from explants of young tortoises but epithelial cells stopped dividing early in the history of all cultures, even at the optimum temperature of 30 °C. Serially subcultured fibroblasts from young tortoises divided more rapidly and achieved longer lifespans than fibroblasts from old tortoises in terms of mean population doublings and to a lesser extent, calendar time. For all cultures the lifespans exceeded those reported for human diploid fibroblasts. The results indicate that a proportionality exists between the potential (remaining) lifespan in vivo and the mitotic capacity of cultured diploid cells in vitro.     

The humoral nature of the colony-stimulating action of bone marrow cells on stromal colony formation in bone marrow cultures

In 24 hours adherent marrow cell cultures (AMCC) were represented by single stretched fibroblasts. In non-feeder-supplemented AMCC most of the CFU-f remained single fibroblasts or passed through 1-3 cell doublings [correction of dudlings]. The colony stimulating activity of irradiated marrow cells was found to be diffuse across the Millipore filter, which seems to indicate that haemopoietic marrow cells produce a colony stimulating factor which is required for triggering the CFU-f from the Go-period of the cell cycle into cell proliferation.     

In vitro immune response of human peripheral lymphocytes. VII. Effect of anti-mu and anti-delta antibodies on B colony formation and detection of abnormal B cells in patients with juvenile rheumatoid arthritis

Effect of anti-mu and anti-delta antibodies on PHA- and protein A-induced B colony formation was studied. Anti-mu antibody at any concentrations tested did not show inhibitory or enhancing effect on colony formation. On the other hand, anti-delta antibody enhanced both PHA- and protein A-induced colony formation. Optimum concentration of anti-delta antibody for maximum enhancement was 0.1 microgram/ml. and F(ab')2 fragment of anti-delta antibody also showed comparable enhancing effect. Simultaneous addition of IgD with anti-delta antibody abrogated anti-delta-induced enhancement, and anti-delta antibody did not show any facilitation of colony formation in IgM+ IgD- cell population. In marked contrast with normal B cells, anti-mu antibody showed a remarkable enhancing effect on protein A-induced colony formation of B cells from JRA patients. F(ab')2 fragment of anti-mu antibody also showed comparable enhancing effect. Anti-mu antibody did not show any enhancement of colony formation of B cells from several other autoimmune diseases. The result indicated the presence of abnormal B cells in JRA patients.     

Synthesis of proline and hydroxyproline in human lung (WI-38) fibroblasts.

Human lung fibroblasts (WI-38) in late exponential phase of growth, in stationary phase after confluency was reached, and at high or low number of population doublings were used to investigate the synthesis of proline and hydroxyproline from glutamate or arginine. Glutamate was from two to five times as effective a precursor as arginine; glutamine did not seem to be involved in these metabolic pathways. Accumulation of protein-bound hydroxyproline in cell layers was observed only after confluency. Confluent cells synthesized more proline from glutamate than did cells in late exponential growth. Conversion of glutamate into intracellular free proline was conducted also to a greater extent in confluent cells at a high number of population doublings. Conversion of glutamate into proline or hydroxyproline in cell-layer protein was not affected significantly by the number of population doublings. Less total protein as well as less hydroxyproline accumulated with cells at a high number of population doublings.     

Mechanism of accessory cell requirement in inducing IL 2 responsiveness by human T4 lymphocytes that generate colonies under PHA stimulation

PHA-driven monoclonal colony formation by low concentrations of resting T4 lymphocytes in agar culture requires the presence of interleukin 2 (IL 2) and accessory cells. Using recombinant IL 2 and anti-Tac monoclonal antibody as a probe for the IL 2 receptor, we demonstrate that the requirement of accessory cells (here an irradiated B cell line) in inducing IL 2 responsiveness relies on their enhancing effect in functional IL 2 receptor expression by the T colony progenitors. Furthermore, it is shown that cell to cell interaction between accessory cells and colony progenitors results in IL 2 response, i.e., colony formation, when the IL 2 receptor density reaches a critical threshold. The asynchronism in IL 2 responsiveness expression by the T colony progenitors upon activation and the short-lived T cell-accessory cell interaction, due to accessory cell death, determine the 10% colony efficiency of the culture system. In addition, we demonstrate that the accessory function in IL 2 receptor and IL 2 responsiveness expression by the T colony progenitors can be supported by irradiated T lymphocytes as well as B cells. The absence of lineage restriction of the signal delivered by accessory cells, and the requirement of physical interaction between T colony progenitors and accessory cells, emphasize the necessity of cross-linking the activation-signal receptors in inducing IL 2 responsiveness by resting T4 cells.