Cycloheximide effect on DNA degradation and delta-crystallin synthesis in terminally differentiating lens cells

Low concentrations of a protein synthesis inhibitor, cycloheximide, were added throughout the process of in vitro differentiation of 11-day old embryonic chick lens cells. We found with low concentrations of cycloheximide (0.01 to 0.03 microgram/ml, 3 days of culture), that there was an almost complete delay of DNA degradation as observed on alkaline sucrose gradient. Identical concentrations and exposure time had no blocking effect on increased delta-crystallin synthesis as detected by immunoprecipitation and electrophoresis. Higher concentrations of cycloheximide (0.1 to 1 microgram/ml) showed a marked effect on DNA size and a net inhibition on delta-crystallin synthesis. Thus a selective effect of low doses of cycloheximide was observed on terminal differentiation suggesting that there was not a relationship between DNA degradation and delta-crystallin synthesis in these short term experiments. The investigations of minor proteins could be of interest as they may have a crucial role in intact nuclei cataracts.     

Increased sensitivity of various genes to endogenous DNase activity in terminal differentiating chick lens fibers

In the lens, epithelial cells from the equatorial zone differentiate into postmitotic elongated fibers. One aspect of this differentiation is nuclear shape transformation and DNA degradation. This process is controlled by DNase activity which in fiber nuclei increases with development. DNase activity is also present in the epithelial cell nuclei which appears to be non-functional but could be activated in vitro by exogenous addition of Ca2+. We have analyzed the possible selective action of endogenous DNase on 3 genes involved in lens terminal differentiation, namely delta-crystallin, beta-tubulin and vimentin, and on 1 gene not thought to participate in this process, ovalbumin. We have compared restriction DNA patterns of these genes in nuclei isolated from 11-day-old chick embryos and incubated in Ca2+-free medium or in fresh epithelial and fiber lens tissue at 11 and 18 days of development. During incubation in vitro of 11-day fiber nuclei, there is a net increase in the sensitivity of the delta-crystallin, beta-tubulin, ovalbumin and vimentin chromatin to the endogenous DNase. The vimentin gene appears to be more stable than the beta-tubulin and delta-crystallin genes indicating a degree of specificity of the endogenous DNase activity. In the epithelial nuclei, the lens-specific genes appear to be more stable but paradoxically there is a net degradation of the ovalbumin gene. In freshly isolated tissues the 4 genes were detected in epithelial and fiber cells at 11 and 18 days. Furthermore, in the mature fibers in which the nuclei were degenerating, the latter genes were still not completely digested.     

DNA Polymerase, DNA Ligase, and Thymidine Kinase Activity in Chicken Lens, Related to DNA X-Ray Lesion Repair

The activity of DNA polymerases α, β, and γ; DNA ligases I and II; and thymidine kinase in chicken lenses is determined. These enzymes are present in embryonic intact lenses freshly isolated at 6 days and 11 days of development and in lenses isolated at 11 days of development and cultured for three days. They are also found in both epithelium and fibers when separated at 10 days of embryonic development and in the epithelium of 14½-month-old hen lenses. In the anucleate mature hen lens fibers, the only detected enzyme is thymidine kinase. Previous results showed that repair of X-ray irradiated DNA was total in 11-day-old embryonic lenses while repair was not detected at six days. On the other hand, repair was very much impaired when the 11-day-old lenses were cultured for three days. Enzymic analyses suggest that the defect in DNA repair and the spontaneous DNA degradation observed in chick lenses at certain embryonic stages, are not due to the absence of any of the above enzymic activities. Alternative explanations are discussed.     

Collagen synthesis by long-lived mRNA in embryonic chicken lens

Lens capsule collagen synthesis by epithelial and fiber cells was examined by immunoprecipitation and collagenase digestion in embryonic and posthatch chicken eye lens. Epithelial cells and lens fibers in the process of terminal differentiation produce alpha 1 and alpha 2 type IV collagen chains. At 6 days of embryonic development in addition to the alpha 1 (IV) and alpha 2 (IV) collagen chains, lens cells produce high molecular weight collagenase-sensitive proteins not immunologically related to type IV collagen. Lens capsule collagen components have been identified in central and outer fibers isolated from 18-day embryos and from 10-day posthatch chicken eyes. At these stages, fibers which have an increasing number of picnotic nuclei still show collagen synthesis due to long-lived mRNA. Analysis of collagen synthesis by lens cells incubated with actinomycin D suggests that stabilization of collagen mRNA occurs in lens fiber cells and to a lesser extent in epithelial cells as early as 6 days of embryonic development.     

Endogenous nuclease activity in chick embryo lens cells

The temporal and spatial sequence of nuclear disappearance during the terminal differentiation of lens fiber cells could be due to an impairment of the DNA repair pathways or to the appearance of an active DNA degradation process. The results presented here favor the second hypothesis. A single-stranded DNA nuclease activity and a double-stranded DNA nuclease activity have been found in chick embryo fiber cells. Moreover, there is a good correspondence between the variations of the nuclease activity and the stages of differentiation of the different samples analyzed.     

Cell proliferation is not required for the initiation of early cleft formation in mouse embryonic submandibular epithelium in vitro

An X-ray irradiation method was employed to analyse the role of cell proliferation in vitro in the cleft formation of mouse embryonic submandibular epithelium at early stages. When the mid 12-day gland was exposed to 200 rad of X-rays, the growth was severely retarded. In contrast, late 12-day and early 13-day glands grew apparently in a normal fashion, as did the control gland, for up to 40 h. In either case, they formed shallow clefts within 10 h of culture. With 1000 rad irradiation, the mid 12-day gland did not grow at all, but formed clefts within 20 h of culture followed by a rapid degeneration. Under the same conditions, the growth of the late 12-day gland, which was at the stage just before branching, was retarded until 10 h of culture, followed by a slight increase in epithelial size, but cleft formation was also observed within 6-10 h, as in the control gland. When exposed to a dose of 1000 rad of X-rays, the early 13-day and the late 12-day glands exhibited similar radiosensitivity; the initial narrow clefts in the epithelium deepened and new clefts began to form within 6-10 h of culture. [3H]thymidine incorporation studies revealed that a dose of 1000 rad reduced DNA synthesis of mid and late 12-day glands by 72 and 65%, respectively. Histological examination of X-irradiated late 12-day gland showed that mitotic figures were rarely seen in the epithelium at 6 h of culture. Aphidicolin, a specific inhibitor of DNA synthesis, could not halt the cleft formation of the late 12-day gland. In this experiment 89% of DNA synthesis was inhibited. Treatment of an X-ray irradiated late 12-day gland with aphidicolin blocked 92% of the DNA synthesis, but did not prevent cleft formation taking place. These results indicate that neither cell division nor DNA synthesis, is required for the initiation process of the cleft formation of the mouse embryonic submandibular epithelium at early morphogenetic stages in vitro.     

Contrasting patterns of c-myc and N-myc expression in proliferating, quiescent, and differentiating cells of the embryonic chicken lens.

The present study uses the polymerase chain reaction and in situ hybridization to examine c-myc and N-myc mRNA in the embryonic chicken lens at 6, 10, 14 and 19 days of development and compares the pattern of expression obtained with the developmental pattern of cell proliferation and differentiation. In the central epithelium, c-myc mRNA levels were proportional to the percentage of proliferating cells throughout development. N-myc mRNA expression in this region was relatively low and showed no correlation with cell proliferation. The ratio of N-myc to c-myc mRNA increased markedly with the onset of epithelial cell elongation and terminal fiber cell differentiation, although both c-myc and N-myc mRNAs continued to be expressed in postmitotic, elongating cells of the equatorial epithelium and in terminally differentiating lens fiber cells. Thus, increased expression of N-myc, a gene whose protein product may compete with c-myc protein for dimerization partners, accompanies the dissociation of c-myc expression and cell proliferation during terminal differentiation of lens fiber cells.     

The synthesis and localization of crystallins in different cell compartments of the crystalline lens in adult frogs: immunoautoradiographic and immunofluorescent research

The purpose of this study was to analyze immunochemically the synthesis and distribution of tissue-specific proteins, i.e., alpha-, beta- gamma- and rho-crystallins, in morphologically distinct regions of the frog (Rana temporaria L.) lens which consist of cells at various stages of differentiation, maturation and aging. Five such cell compartments can be distinguished in the lens: (1) central zone of lens epithelium (stem/clonogenic cells); (2) equatorial epithelial cells (differentiating cells); (3) lens fibers of the outer cortex (post-mitotic differentiated cells); (4) lens fibers of the deep cortex (cells without nuclei at terminal stage of differentiation); and (5) cells of the lens "nucleus" (cells formed during embryogenesis). Intact lenses and isolated lens epithelium were cultured in vitro in the presence of 35S-methionine. Then lens epithelium, outer and deep cortex, and lens nucleus were extracted with buffered saline and extracts used for immunoautoradiography. Distribution of crystallins in paraffin sections of the whole lens or isolated lens epithelium was studied using indirect immunofluorescence. Synthesis of alpha-crystallins was observed in lens epithelium and cortex, but not in lens nucleus. According to immunohistochemistry, these proteins were absent from central part of the lens epithelium: positive fluorescence was observed only in elongating cells at its periphery and in lens fibers. The data on beta-crystallins are similar except that synthesis of these proteins (traces) was detected also in lens nucleus. Synthesis of gamma-crystallins was detected in lens cortex and nucleus (traces) but not in epithelium. Immunohistochemistry showed that these proteins are absent from all regions of lens epithelium and found only in fiber cells of cortex and nucleus. Rho-crystallin was synthesized in all cell compartments of the adult lens, and all lens cells contained this protein. Our results show that cells of central lens epithelium do not contain alpha- beta- or gamma-crystallins (or the rate of their synthesis is insignificant). While cells are moving towards lens equator and elongating, synthesis of alpha- and beta-crystallins is activated. Gamma-crystallins are synthesized later, first in young lens fibers near lens equator. During embryonic development in amphibia, in contrast, gamma- and beta-crystallins are detected at earlier stages than alpha- and rho-crystallins (Mikha?lov et al., 1988). These data suggest that different mechanisms are involved in differentiation on lens fibers from embryonic precursor cells, on one hand, and from epithelial stem cells of adult lens, on the other.     

DNAase activities in embryonic chicken lens: in epithelial cells or in differentiating fibers where chromatin is progressively cleaved.

Lens is an organ composed of a layer of epithelial cells and a mass of fibers. During terminal differentiation, epithelial cells from the equatorial region elongate into fibers, nuclei change shape, the chromatin appears much condensed in the last step of differentiation and the DNA breaks down into nucleosomes. The pattern of DNAase activities has been recorded at different chick embryonic stages (11 and 18 days) using polyacrylamide gel electrophoresis with DNA substrate in the gel matrix. Two DNAases (30 and 40 kDa) have been observed in lens epithelia and fibers at both stages. However, the activities of both of the enzymes are augmented in fiber cells. The 30 kDa DNAase requires and Ca2+ and Mg2+ (5-15 mM) to hydrolyze the DNA substrate while the 40 kDa-activity is inhibited by added divalent cations (5-15 mM). The 30 kDa protein is inhibited by Na+ and is probably an endonuclease. Both nuclease activities probably are involved in the cleavage of fiber chromatin into nucleosomes during lens terminal differentiation, but variables such as chromatin configuration, unmasked DNA sequences, presence of cations, and pH gradients probably determine the extent of involvement of each DNAase.     

TOPOGRAPHY OF DNA SYNTHESIS IN THE MAMMARY GLAND OF THE C3H MOUSE AND ITS CONTROL BY OVARIAN HORMONES: AN AUTORADIOGRAPHIC STUDY

Tritium-labelled thymidine was injected 45 min before sacrifice into virgin female C3H/HeJ mice 7–23 weeks of age, as well as into 10-week-old mice which had been ovariectomized and treated daily with 1 μg of oestradiol-17β and/or 1 mg of progesterone. Autoradiographs were made of squash preparations of the mammary glands, stained by Feulgen's method. The following results were obtained: (1) During normal development of the gland, cells synthesizing DNA are abundant in terminal buds and virtually absent in duct epithelium. Hence ductal growth takes place by the addition of cells produced in the terminal end structures. (2) At 5–6 months, when mammary growth has ceased, a considerable number of cells synthesizing DNA can still be found in alveoli, though not in duct epithelium. Hence the alveolar cells constitute a renewal population in the adult virgin. Because they maintain the potentiality to divide, duct cells are a G₀ population. (3) Ovariectomy results in arrest of DNA synthesis within 3–5 days. Both oestradiol and progesterone restore DNA synthesis in alveoli but only progesterone is able to induce DNA synthesis in duct epithelium, and the differentiation of terminal buds into alveoli. This finding provides an explanation for the resumed proliferation of duct cells in pregnancy. (4) The number of cells engaged in DNA synthesis varies considerably among identical structures within the same gland. This may be due either to synchrony of cell replication and/or to fluctuations of proliferative activity in the gland.     

DIFFERENTIATION AND DEDIFFERENTIATION OF RAT LENS EPITHELIAL CELLS IN SHORT- AND LONG-TERM CULTURES

The crystallin synthesis of rat lens cells in cell culture systems was studied in relevance to their terminal differentiation into lens fibers. SDS-gel electrophoresis combined with several immunological techniques showed that γ-crystallin is a fiber-specific lens protein and is not localized in the epithelium of either newborn or adult lenses. When lens epithelial cells of newborn rats were cultured in vitro , α-crystaIlin was detected in many, but not all, of cells cultured for 10 days. Cells with α-crystallin gradually changed their shape into a flattened filmy form and finally differentiated into lentoid bodies. The differentiation of lentoid bodies was also found in cultures of epithelial cells obtained from adult lenses. The molecular constitution of lentoid bodies was the same as that of lens fibers in situ . The differentiation of lentoid bodies occurred successively for 5 months in cultures of lens epithelial cells. Most of the proliferating cells, however, lost α-crystallin during the culture period. Thereafter, they did not show any sign of further differentiation into lens fibers. Four clonal lines were established from these cells. One protein which is specific to the lens epithelium and the neural retina in situ (tentatively named as β_u-crystallin) was maintained in all lines, suggesting that some specific properties of ocular cells remain in the lined cells.     

Human papillomavirus and cervical cancer

Human papillomavirus (HPV) is a small non-enveloped icosahedral virus with a circular double-stranded DNA genome of 8 kilo base pairs. HPV particles reach and infect the basal cells of the stratified epithelia through small epithelial lesions. In the basal cells the viral DNA is maintained as episomes, which start to replicate when the host cells initiate terminal differentiation. In these differentiating cells the degradation of p53 by the E6 protein and the abrogation of the pRb functions by the E7 protein lead to the reactivation of the DNA synthesis machinery. After virus propagation the host cells usually die. On the other hand, in some of the infected cells, the E6 and E7 genes are integrated on rare occasion into cell DNA. The cell continuously expressing the E6 and E7 proteins from the integrated genes is immortalized and sometimes acquires malignant phenotype induced by the accumulated damages to DNA. Of more than 100 HPV genotypes recorded to date, 13 including types 16 and 18 are associated with cervical cancer. Expression of HPV major capsid protein L1 in some cultured cells results in production of virus-like particles (VLPs). The VLPs of types 6, 11, 16, and 18 were used as a prophylactic vaccine in recent clinical trials and shown to successfully induce type-specific neutralizing antibodies in the recipients.     

Developmental decline in DNA repair in neural retina cells of chick embryos: persistent deficiency of repair competence in a cell line derived from late

Neural retinas of 6-day-old chick embryos synthesize DNA and are able to carry out DNA excision repair. However, in contrast to the situation in human cells, the maximum rate of repair induced by N-acetoxy acetylaminofluorene (AAAF) is no greater than that induced by methyl methanesulfonate (MMS). With advancing differentiation of the retina in the embryo, cell multiplication and DNA synthesis decline and cease, and concurrently the cells lose the ability to carry out DNA excision repair. Thus, in 15-16-day embryos, in which the level of DNA synthesis is very low, DNA repair is barely detectable. If retinas from 14-day embryos are dissociated with trypsin and the cell suspension is plated in growth- promoting medium, DNA synthesis is reinitiated; however, in these cultures there is no detectable repair of MMS-induced damage, and only low levels of repair are observed after treatment with AAAF. A cell line was produced, by repeated passaging of these cultures, in which the cell population reached a steady state of DNA replication. However, the cell population remained deficient in the ability to repair MMS-induced damage. This cell line most likely predominantly comprises cells of retino-glial origin. Possible correlations between deficiency in DNA repair mechanisms in replicating cells and carcinogenesis in neural tissues are discussed.     

Juvenile hormone regulation of structural changes and DNA synthesis in the follicular epithelium of Leucophaea maderae

Juvenile hormone (JH I) stimulates specific morphological and biochemical changes in the follicular epithelium surrounding the terminal oöcytes in Leucophaea maderae. These include extracellular and intracellular structural changes, increased rates of follicle cell DNA synthesis, and elevated follicle cell DNA concentrations.Using females decapitated 24 hr after ecdysis, we have shown that JH I injections stimulate the following structural changes in the follicular epithelium: the appearance of channels between adjacent follicle cells and of spaces between the follicular epithelium and the maturing oöcyte; an increase in follicle cell size; the development of an extensive rough endoplasmic reticulum system; and an enlarged nucleus within each follicle cell. No increase in the number of follicle cells surrounding the developing terminal follicles is found in 7-day JH I-treated females, although the terminal follicles are almost twice as long as those in untreated females.In addition, we have demonstrated that JH stimulates the following biochemical events in the ovary: a 3.5 fold increase in thymidine incorporation into follicle cell DNA, with no subsequent transfer of such DNA to the developing oöcyte, and a 1.4 fold increase in ovarian DNA in 7-day JH-treated females. These data indicated that JH stimulates follicle cell DNA synthesis. The absence of any corresponding division of follicle cells suggests that JH I may induce polyploidy in follicle cells.Extended exposure of decapitated females to JH I does not result in complete ovarian maturation. Although fat bodies in the treated insects continue to display an increasing rate of vitellogenin synthesis, DNA synthesis in the terminal follicles declines rapidly after day 9, and the terminal follicles ultimately degenerate.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

DNA synthesis and repair in terminally differentiating embryonic lens cells

In this study, we have investigated the correlation between in vitro differentiation and DNA degradation. We have shown a decrease in high molecular weight DNA in 10-day-old embryonic chick lens cells after 1 day in culture. Using X-ray as a tool to check the ability of these cells to repair single-strand breaks in their DNA, we have observed, in addition to spontaneous DNA degradation, an impairement in the repair process.     

Repair of the radiation damage in hematopoietic stem cells from the mouse embryonic liver. Kinetic aspects]

The method of fractionated irradiation was used to study kinetic aspects of repair of sublethal radiation damages in precursor cells from mouse embryonal liver that form in vivo colonies on 8th and 11th days. It was shown that 11-day CFUs had a lesser ability to repair sublethal radiation damages than 8-day ones at different time-intervals between radiation fractions (from 2 to 6 h). These two CFUs sub-populations differed also in the repair kinetics.