A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells.

To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.

Effect of estrogen-promoted bacterial infections of the rat uterus on bioassay of mammalian cell growth factor activities in uterine luminal fluid.

Exogenous estradiol treatment of intact or ovariectomized rats causes accumulation of significant volumes of fluid in the uterine horns. In this report, evidence is presented showing the presence of mammalian cell growth factor(s) in uterine luminal fluid (ULF), along with other data showing that the exogenous estradiol treatment needed to cause significant accumulation of the fluid also facilitates the movement of vaginal origin bacteria into the uterine horns. It is shown that microorganisms infect the uteri of 80% or more of rats administered exogenous estradiol, and that the microorganisms are most probably of vaginal origin; procedures such as ligation of the uterine body above the cervix or antibiotic treatment did not suppress the infections. Administration of different doses of exogenous estrogen by either implantation of a single 25-mg estradiol/cholesterol pellet which causes a 20- to 50-fold elevation of estradiol levels above physiological plasma concentrations, or instead, by a Silastic tube delivery method that elevates levels only 2- to 3-fold above the normal range, resulted in equal frequency of uterine infections and in the appearance of infection at the same time after starting treatment. A number of bacterial species are present in the contaminated ULF, and these are the origins of intracellular products which are potent inhibitors of mammalian cell growth; the presence of these bacterial origin inhibitors interferes with the bioassay of the ULF growth factor activity, and hence, impedes the characterization of the growth factor(s) present in luminal fluid. Characterization of the origins of the growth-inhibiting activities showed that Pseudomonas aeruginosa and Proteus mirabilis are the predominant species present in infected uteri and that both produce exotoxin activities which inhibit growth of mammalian cells in culture; Pseudomonas appears to be the greater producer of cytotoxic activity. Evidence is presented that suggests that the well-known Exotoxin A produced by Pseudomonas may be responsible, in part, for the toxic effects of this organism. Other, as yet unidentified, cell growth inhibitors also may be produced by the bacteria found in ULF. Surgical separation of the uterine body from the cervix allows preparation of ULF which contains no bacteria and substantially reduced levels of growth inhibitors to mammalian cell lines.

Identification of estrogen-inducible growth factors (estromedins) for rat and human mammary tumor cells in culture.

The role of polypeptide growth factors (estromedins) as mediators of estrogen-responsive mammary tumor growth is studied in this report. Three possible new mechanisms were investigated that include endocrine, autocrine, and paracrine related growth factors. The first hypothesis being tested is whether estrogens interact with target tissues and cause the biosynthesis and secretion of polypeptide growth factors, which then act as mitogens for normal and neoplastic mammary tissues. Data presented suggest that this mechanism involves estrogen interaction with uterus, kidney, and pituitary gland causing production of growth factors, which then enter the general circulation and promote growth of distant target tissues. This is an endocrine type mechanism. Another type of estromedin control (autocrine control) may be exerted in an autostimulatory way in which the target tissue produces the polypeptide factors for its own growth in response to estrogen stimulation. A variation of the autocrine mechanism may be a paracrine mechanism in which some cells of an estrogen-responsive normal or neoplastic tissue produce growth factors that act on adjacent or neighboring cells. From the data available, all three possible types of growth factors could be functioning synergistically to yield the final result of continuous estrogen responsive tumor growth in vivo.

Properties of a growth factor activity present in crude extracts of rat uterus.

We have shown previously (D. A. Sirbasku, 1978, Proc. Natl. Acad. Sci. U.S.A., 75:3786-3790) that an estrogen-inducible growth factor activity for rat mammary and rat pituitary tumor cells can be identified in extracts of rat uteri, although at the time of that report only a limited biochemical characterization of the activity was presented. In this report, we have evaluated the growth factor activity for lipid, steroid hormone or protein-like properties. Uterine growth factor activity was assayed by measure of the increased cell number of the MTW9/PL rat mammary tumor cell line established by this laboratory and described previously (D. A. Sirbasku, 1978, Cancer Res. 38:1154-1165). Studies showed the following characteristics of growth factor activity: destroyed by trypsin treatment; labile when heated at 80 degrees C; partially denatured by 6 M guanidine or 8 M urea treatment or 50% aqueous solutions of organic solvents; inactivated by extremes of pH or overnight treatment with mild acid; not dialyzable at neutral pH; of apparent molecular weight of 70,000 daltons by G-100 Sephadex chromatography; possessing an isoelectric point of 4.8 to 5.2; not chloroform/methanol extractable; and not in any way identified as either a lipid or a steroid hormone. The data available suggest that the uterine growth factor activity is a protein or polypeptide of apparent high molecular weight, and that the activity does not directly correspond to other known growth factors.