Roobik Azarnia

Autotransplantation of Retinal Pigment Epithelium in Intravitreal Diffusion Chamber

Retinal pigment epithelial (RPE) cells were autotransplanted intravitreally in diffusion chambers in rabbit eyes. The RPE cells underwent transformation; first into macrophages, later into spindle-shaped cells with collagen production having epithelial cell characteristics. DNA replication, shown by autoradiography, and loss of pigment granules indicated proliferation of these cells. These results support the hypothesis that RPE proliferation and transformation play a major role in the formation of collagen containing membranes, such as are found in massive periretinal proliferation.

Intercellular communication and the control of growth: X. Alteration of junctional permeability by thesrc gene. A study with temperature-sensitive mutant Rous sarcoma virus

SummaryTo study changes of junctional membrane permeability associated with transformation, the junctions and the nonjunctional membranes of quail embryo-, chick embryo- and mouse-3T3 cell cultures, infected with temperature-sensitive mutant Rous sarcoma virus, were probed with fluorescent-labelled glutamate. Junctional permeability fell in the transformed state. In the quail cells, the fall was detectable within 25 min of shifting the temperature down to the level (permissive) at which tyrosine-phosphorylation by the viralsrc gene product is expressed. This reduction of junctional permeability is one of the earliest manifestations of viral transformation. Normal permeability was restored within 30 min of raising the temperature to the nonpermissive level, a reversibility that could be displayed several times during the span of a cell generation. The reversal seems to reflect a reopening of cell-to-cell channels rather than a synthesis of new ones; it is not blocked by protein-synthesis inhibition. Treatments with cyclic AMP and phosphodiesterase inhibitor or with forskolin, which stimulate serine and threonine phosphorylation—the type of phosphorylation on which normal junctional permeability depends (Wiener & Loewenstein, 1983,Nature 305∶433)—did not abolish, in general, the junctional effect of the virus;src tyrosine-phosphorylation apparently overrides the junctional upregulation mediated by cyclic AMP. Nonjunctional membrane permeability was not sensibly affected by the virus. It was affected, however, by temperature: lowering the temperature from the nonpermissive to the permissive level caused the nonjunctional permeability to fall, andvice versa. This change was unrelated to transformation. Its secondary effect on junctional transfer is in the opposite direction to that produced by the temperature-activated viral transformation.

Cell junction and cyclic AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type

SummaryThe cyclic nucleotide effect on junction was studied in C1-1D cells, a mouse cancer cell type that fails to make permeable junctions in ordinary confluent culture. Upon administration of cyclic AMP, dibutyryl cyclic AMP, dibutyryl cyclic AMP plus caffeine (db-cAMP-caffeine), or cholera toxin (an adenylate cyclase activator), the cells acquired permeable junctions; they became electrically coupled and transferred fluorescent tracer molecules among each other—a transfer exhibiting the molecular size limit of permeation of normal cell-to-cell channels. The effect took several hours to develop. With the db-cAMP-caffeine treatment, junctional permeability emerged within two hours in one-fifth of the cell opopulation, and within the next few hours in the entire population. This development was not prevented by the cytokinesis inhibitor cytochalasin B. Permeable junctions formed also in two other conditions where the cell-endogenous cyclic AMP level may be expected to increase: serum starvation and low cell density. After three weeks of starving the cells of serum, a junctional permeability arose in confluent cultures, which on feeding with serum disappeared within two to three days. At low cell density, namely below confluency, the cells made permeable junctions, unstarved. In cultures of rather uniform density, the frequency of permeable junctions was inversely related to the average density, over the subconfluent range; at densities of about 1×104 cells/cm2, where the cells had few mutual contacts, 80% of the pairs presumed to be in contact were electrically coupled. In cultures with adjoining territories of high (confluent) and low cell density, there was coupling only in the last, and in this low-density state the cells were also capable of coupling with other mammalian cell types (mouse 3T3-BalbC and human Lesch-Nyhan cells).Correlated electron microscopy of freeze-fractured cell junctions showed no membrane differentiation in confluent C1-1D cultures. The junctions acquired differentiations, namely particle clusters of gap junction and strands of tight junction, upon cyclic nucleotide application or serum starvation and in the lowdensity condition. With db-cAMP-caffeine, these differentiations appeared within 4 hr of the treatment (confluent cultures), growing in size over the next hours. Treatment with cycloheximide, but not with cytochalasin B, prevented the development of recognizable gap junction and tight junction in cultures supplied with db-cAMP-caffeine.

Translation and functional expression of cell-cell channel mRNA in Xenopus oocytes.

SummarymRNA from estrogen-stimulated rat myometrium, a tissue known to upregulate cell-cell channels in response to this hormone, was microinjected intoXenopus laevis oocytes. The oocytes had been freed from covering layers of follicle cells and vitelline to allow direct cell membrane interactions when paired. About 4 hours after the mRNA injection, paired oocytes become electrically coupled. This coupling was due to the presence of typical cell-cell channels characterized by size-limited intercellular tracer flux, the presence of gap junctions at the oocyte-oocyte interface, and the reversible uncoupling that occurred in the presence of carbon dioxide. The induction of new cell-cell channels in the oocyte membrane was observed against a zero background or a low level of endogenous coupling, depending on the maturation stage of the oocytes. The time course of development of cell-cell coupling after the microinjection of mRNA was determined. The mRNA capable of inducing cell-cell coupling was confined to an intermediate size class when fractionated on a sucrose gradient.

Intercellular Communication and Tissue Growth: VIII. A Genetic Analysis of Junctional Communication and Cancerous Growth

SummaryNormal, proliferating cells are interconnected at their junctions by membrane channels through which molecules can pass from cell to cell (Loewenstein, W.R. 1966.Ann. N.Y. Acad. Sci.137:708). A channel-competent, normally growing cell (human fibroblast) was hybridized with a channel-incompetent cancer cell (mouse L-1d cell), and the segregant hybrid clones were analyzed in a genetic approach to the question of whether the junctional membrane channels are instrumental in transmission of growth-controlling molecular signals. The channel competence of the human parent was characterized by the ability to transfer small inorganic ions (electrical coupling) and fluorescein, and the growth patterns of this cell, by growthin vitro to low saturation densities and nontumorigenicity in immuno-suppressed hosts. The mouse parent cell had the opposite characteristics. The early hybrid generations (which still had a large part of each parent chromosome complement) were of two classes: one class resembled the human parent cell in channel competence,in vitro growth pattern, and low tumorigenicity within 26 days; the other class presented an intermediate expression of channel competence characterized by transfer of small inorganic ions but not of fluorescein. As the hybrid generations lost human chromosomes, there was segregation of several biochemical and morphological traits, but no segregation of channel competence and normal growth traits. Among the segregants were 22 clones which had reverted to the channel-incompetent trait of the mouse parent. In every case, reversion to the channel defect went hand in hand with reversion to the growth defect, just as, in the early-generation hybrids, correction of the channel defect went hand in hand with correction of the growth defect. Thus, the human genetic factor that corrects the channel defect of the mouse parent cell seems closely linked, if not identical, with that correcting the growth defect. This genetic correlation encourages us in the belief that the channel defect may be an etiological factor in this particular cancer form.

Intercellular communication and tissue growth. VI. Failure of exchange of endogeneous molecules between cancer cells with defective junctions and noncancerous cells.

SummaryThree cancer cell strains that fail to make permeable membrane junctions were tested for ability to transfer an endogenous hypoxanthine derivative from cell to cell. The cells of these strains, loaded with3H-hypoxanthine, were grown in contact with cells from a mutant line incapable of incorporating exogenous hypoxanthine. The transfer of the3H-hypoxanthine derivative to the mutant cells was determined by radio-autography and, in the same preparations, the presence of permeable membrane junctions was determined by intercellular fluorescein tracer diffusion and electrical measurement. The cells of the three strains showed no transfer of hypoxanthine derivative to contiguous mutant cells; the cells that make permeable junctions did show such transfer, under the same conditions.In contrast to this contact-requiring mode of transfer, a contact-independent transfer phenomenon was observed with these three cancer cell strains.

Intercellular communication and tissue growth: IX. Junctional membrane structure of hybrids between communication-competent and communication-incompetent cells.

SummaryThe structure of the membrane junctions of the hybrid cell system, examined in the companion paper in respect to competence for communication through cell-to-cell membrane channels, is here examined by freeze-fracture electron microscopy. The junctions of the channel-competent parent cell and of the channel-competent hybrid cells present aggregates of intramembranous particles typical of “gap junction”; those of the channel-incompetent parent cell and channel-incompetent segregant hybrid cells do not. Competence for junctional communication and for gap junction formation are genetically related. The junctions of the intermediate hybrid cells with incomplete channel-competence (characterized by cell-to-cell transfer of small inorganic ions but not of fluorescein), present special intramembranous fibrillar structures instead of discrete gap-junctional particles. The possibility that these structures may constitute coupling elements with subnormal permeability is discussed in terms of incomplete dominance of the genetic determinants of gap junction.

Intercellular communication and the control of growth: XII. Alteration of junctional permeability by simian virus 40. roles of the large and smallT antigens

SummaryWe studied the action of temperature-sensitive mutant simian virus 40—a transformation-inducing DNA virus—on the junctional permeability to mono-, di- and triglutamate in rat embryo-, pancreas islet (epithelia)-, and 10T1/2 cell cultures. Junctional permeability was reduced (reversibly) in the transformed state. To dissect the genetics of this alteration, we used two kinds of mutant virus DNA. One kind had a temperature-sensitive mutation on theA gene, rendering the largeT antigen (the gene product) thermolabile (T+ ⇆T−). The other had a deletion on theF gene, in addition, abolishing (permanently) the expression of the littlet antigen (t−). The junctional alteration occurred in the conditionT+t+, but not in the conditionsT−t+,T+t− orT−t−. Both antigens, thus, are necessary for this junctional alteration—a genetic requirement identical to that for decontrol of growth (but distinct from that of the cytoskeletal alteration).

Intercellular Communication and Cancer

Among the several different forms of cell interactions known to exist in multicellular organisms intercellular communication provides the most direct form of communication by allowing free exchange of ions and small molecules between neighboring cells via permeable membrane junctions. This type of communication has been implicated in various biological functions such as cell differentiation, embryonic development, and tissue growth regulation. In this chapter we will review highlights of functional and structural evidence of junctional communication in normal and cancerous tissues and discuss the existing hypotheses concerning the possible significance of cell junctions in cancer.

Intercellular communication and tissue growth: VII. A cancer cell strain with retarded formation of permeable membrane junction and reduced exchange of a 330-dalton molecule

SummaryA cancer (hepatoma) cell strain is described in which the formation of junctional membrane channels is abnormally slow. The development of electrical junctional coupling following the establishment of contact between these (reaggregated) cells is at least 15 times slower than that between their normal counterparts; and junctional transfer of fluorescein eventually develops, but only in about 5% of the contacts (as against 100% normally). This deviant membrane behavior is interpreted as a retardation in the process of accretion of junctional membrane channels. Its possible etiological role in defective growth regulation is discussed.