Translocation of Src kinase to the cell periphery is mediated by the actin cytoskeleton under the control of the Rho family of small G proteins.

We have isolated Swiss 3T3 subclones that are resistant to the mitogenic and morphological transforming effects of v-Src as a consequence of aberrant translocation of the oncoprotein under low serum conditions. In chicken embryo and NIH 3T3 fibroblasts under similar conditions, v-Src rapidly translocates from the perinuclear region to the focal adhesions upon activation of the tyrosine kinase, resulting in downstream activation of activator protein-1 and mitogen-activated protein kinase, which are required for the mitogenic and transforming activity of the oncoprotein. Since serum deprivation induces cytoskeletal disorganization in Swiss 3T3, we examined whether regulators of the cytoskeleton play a role in the translocation of v-Src, and also c-Src, in response to biological stimuli. Actin stress fibers and translocation of active v-Src to focal adhesions in quiescent Swiss 3T3 cells were restored by microinjection of activated Rho A and by serum. Double labeling with anti-Src and phalloidin demonstrated that v-Src localized along the reformed actin filaments in a pattern that would be consistent with trafficking in complexes along the stress fibers to focal adhesions. Furthermore, treatment with the actin-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, prevented v-Src translocation. In addition to v-Src, we observed that PDGF-induced, Rac-mediated membrane ruffling was accompanied by translocation of c-Src from the cytoplasm to the plasma membrane, an effect that was also blocked by cytochalasin D. Thus, we conclude that translocation of Src from its site of synthesis to its site of action at the cell membrane requires an intact cytoskeletal network and that the small G proteins of the Rho family may specify the peripheral localization in focal adhesions or along the membrane, mediated by their effects on the cytoskeleton.

Bystander effects of different enzyme-prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes.

Transfer of suicide genes into tumor cells renders them sensitive to cytotoxic effects of specific prodrugs. We show here that both the herpes simplex virus thymidine kinase/ganciclovir (tk/GCV) and thymidine phophorylase/5'-deoxy-5-fluorouridine (tp/DFUR) suicide gene systems can induce cell death in tumor cells. Additionally in mixed cultures of cells with and without the suicide gene, death occurred in both cell types, indicative of a bystander effect. We demonstrate, in human and rodent cell lines, that the tk/GCV bystander effect requires gap junctional intercellular communication (GJIC). Where cultures lack GJIC, no bystander effect was observed. In communicating cultures, no correlation between level of GJIC and bystander effect was seen and this was due to high levels of tk activity. Additionally, we demonstrate that transfer of toxic metabolites from tk+ to tk- cells occurs within 2 hr of GCV application and, as no apoptosis could be detected until after this time, apoptosis is the result, not the cause, of the tk/GCV bystander effect. In the tp/DFUR system, a medium-mediated bystander effect, independent of GJIC and apoptosis, was observed. We demonstrated that combining tk/GCV and tp/DFUR suicide gene systems in culture was more effective than either therapy alone.

The bystander effect of the nitroreductase/CB1954 enzyme/prodrug system is due to a cell-permeable metabolite.

The bystander effect is an important part of tumor kill using gene-directed enzyme prodrug therapy (GDEPT). Recently, we have described a novel enzyme prodrug system using bacterial nitroreductase and the prodrug CB1954 (NTR/CB1954). We demonstrate here the presence of a cell-permeable cytotoxic activity in the conditioned growth medium of nitroreductase (NTR)-transduced cells treated with CB1954 and show that its appearance corresponds to the appearance of two metabolites of CB1954 previously identified (Friedlos et al., 1992). The degree of bystander effect and the degree of transferred cytotoxicity correlates with the level of NTR enzyme expression. Two other prodrugs for NTR show little bystander killing and do not produce detectable cell permeable metabolites. The elucidation of the mechanism of the bystander effect may allow the more effective use of NTR/CB1954.

Patterns of junctional communication in skin.

The patterns of junctional communication in whole skin have been studied by iontophoretic injection of the fluorescent dye Lucifer Yellow CH into excised strips of tissue from newborn (less than 48-h) mice. Sections of injected specimens embedded in resin show that: coupling in the dermis is extensive, the injected dye spreads to more than 500 cells in 5 min; coupling in the epidermis is more restricted, dye spreads to less than 25 cells in 5 min; cells of the epidermis are not detectably dye-coupled to the cells of the dermis except at occasional restricted sites, probably related to hair follicle development; immature sebaceous gland cells are coupled but appear to be isolated from other cell types; and endothelial cells in the dermis are coupled to surrounding fibroblasts.

DNA repair in cells from patients with actinic keratosis.

UV-induced DNA repair was studied in peripheral blood lymphocytes from patients with multiple actinic keratoses (AK) requiring surgical therapy and from age-matched normal control individuals. The DNA repair activity in lymphocytes from AK patients at 4 h after UV-irradiation was 50% of that in control lymphocytes, but at 21 h the extent of DNA repair synthesis was similar to that in control cells.

Interactions between normal mammary epithelial cells and mammary tumour cells in a model system.

Normal mammary epithelial (NME) cells and MCF-7 cells aggregate and grow as spheroids when cultured on extracellular matrix derived from Engelbreth/ Holmes/Swarth (EHS) tumour. NME cells stop dividing and differentiate but MCF-7 cells continue to proliferate, although growth is counterbalanced by cell death. In mixed cultures of NME cells and MCF-7 cells, the two cell types form mixed aggregates but then segregate to form well separated domains, often joined by only a narrow neck of cells. In these mixed cultures the growth of MCF-7 cells is inhibited by a factor secreted by NME cells into the medium.

Some murine thymic lymphocytes can form gap junctions.

Analysis of thymic lymphocytes isolated from weanling mice has revealed a minority population able to form permeable, intercellular (gap) junctions. This population is largest in mice aged between 3 and 6 weeks, much smaller in fetal and new-born mice and undetectable in mice aged 12 weeks or more. Fractionation of the thymocytes on Percoll gradients or with peanut agglutinin (PNA) shows the cells able to form junctions are enriched in lower density fractions and agglutinated by PNA, suggesting they are among the most immature. Fractionation by complement mediated cytotoxicity (CMC) and by fluorescence activated cell sorting (FACS) using monoclonal antibodies to specific cell surface determinants shows the junction forming cells are Lyt-1+/Lyt-2- and that the phenotype is associated with both high and low Thy-1 and H-2K epitope densities.

Autofluorescence characteristics of immortalized and carcinogen-transformed human bronchial epithelial cells.

Tissue autofluorescence has been explored as a potential method of noninvasive pre-neoplasia (pre-malignancy) detection in the lung. Here, we report the first studies of intrinsic cellular autofluorescence from SV40 immortalized and distinct tobacco-carcinogen-transformed (malignant) human bronchial epithelial cells. These cell lines are useful models for studies seeking to distinguish between normal and pre-neoplastic human bronchial epithelial cells. The cells were characterized via spectrofluorimetry and confocal fluorescence microscopy. Spectrofluorimetry revealed that tryptophan was the dominant fluorophore. No change in tryptophan emission intensity was observed between immortalized and carcinogen-transformed cells. Confocal autofluorescence microscopy was performed using a highly sensitive, spectrometer-coupled instrument capable of limiting emission detection to specific wavelength ranges. These studies revealed two additional endogenous fluorophores, whose excitation and emission characteristics were consistent with nicotinamide adenine dinucleotide (NADH) and flavins. In immortalized human bronchial epithelial cells, the fluorescence of these species was localized to cytoplasmic granules. In contrast, the carcinogen-transformed cells showed an appreciable decrease in the fluorescence intensity of both NADH and flavins and the punctate, spatial localization of the autofluorescence was lost. The observed autofluorescence decrease was potentially the result of changes in the redox state of the fluorophores. The random cytoplasmic fluorescence pattern found in carcinogen-transformed cells may be attributed to changes in the mitochondrial morphology. The implications of these results to pre-neoplasia detection in the lung are discussed.

In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy.

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)-induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 +/- 25 nm emission band relative to the fluorescence intensity integrated over the entire 400-600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.

Structure of the ductin channel.

Gap junctions appear to be essential components of metazoan animals providing a means of direct means of communication between neighboring cells. They are sieve-like structures which allow cell-cell movement of cytosolic solutes below 1000 MW. The major role of gap junctions would appear to be homeostatic giving rise to groups of cells which act as functional units. Ductin is the major core component of gap junctions and recent structural data shows it to be a four alpha-helical bundle which fits particularly well into a low resolution model of the gap junction channel. Ductin is also the main membrane component of the vacuolar H+-ATPase that is found in all eukaryotes and it seems likely that the gap junction channel first evolved as a housing for the rotating spindle of these proton pumps. Because ductin protrudes little from the membrane, other proteins are required to bring cell surfaces close enough together to form gap junctions. Such proteins may include connexins, a large family of proteins found in vertebrates.

Inhibition and recovery of DNA synthesis in PHA-stimulated u.v.-irradiated lymphocytes from patients with actinic keratosis.

The recovery, after inhibition by u.v.-irradiation, of phytohaemagglutinin-stimulated DNA synthesis is impaired in lymphocytes from patients with multiple actinic keratoses (AK) compared to the recovery in lymphocytes from age-matched, control individuals. This shows that the reduced level (50%) of DNA repair activity in AK cells is sufficiently different from that in normal cells to significantly affect cellular activity.

DNA repair deficiency in lymphocytes from patients with actinic keratosis.

DNA repair activity was measured in peripheral blood lymphocytes from 18 patients with Actinic Keratosis and 18 age-matched control subjects, by comparing the incorporation of 3H-thymidine into cells after irradiation with ultraviolet light with that into unirradiated cells. The incorporation was followed autoradiographically or by measuring acid insoluble radioactivity in cells labelled in the presence of hydroxyurea. The repair activity in lymphocytes from Actinic keratosis patients was only 47.1 percent (+/- 6.5%) of that in cells from the control subjects.

Obstetric outcomes in a rural family practice: an eight-year experience.

There has been debate in some quarters of whether family physicians should do obstetrics and of whether rural hospitals should provide obstetric services. Forks, Washington, is a remote logging town where family physicians and midlevel practitioners have been the sole providers of labor and delivery services. Forks offers an opportunity to evaluate the quality of an isolated rural family practice obstetric service. A retrospective audit of all labor and delivery patient charts at Forks Community Hospital from 1975 to 1983 was undertaken; 1,052 charts were abstracted with 36 factors of morbidity, mortality, and intervention examined. The results, when compared with similar studies in the literature, provide evidence of good performance. In addition, a relatively high-risk obstetric population was served with favorable outcomes. Family physicians and rural hospitals can provide high-quality obstetrical services.

The gap junctional channel.

Two distinct forms of intercellular communication have been found in animal tissues, one using the familiar, trans-membrane, extracellular route and the other using an entirely intracellular route. The intracellular route depends on specialized, permeable (gap) junctions which form at areas of contact between adjacent cells. The junctions contain aqueous channels which directly link the cytoplasms of the coupled cells. Small ions and molecules pass through these channels and move freely between all cells in coupled populations. The structural protein which forms the gap junctional channel has been isolated and characterized. It has an apparent M.Wt. of 16,000 and readily forms multimeric structures. In the membrane, six protein subunits surround the central aqueous pore. Addition of retinoic acid to cells appears to close the junctional channels. This effect of retinoic acid on the junctional pathway of intercellular communication may explain some of its biological activities.

The role of junctional communication in animal tissues.

Permeable intercellular junctions are a common feature of most animal tissues. These junctions allow the free exchange of small ions and molecules between all the cells in coupled populations. Such limited syncytial interaction contributes to the integration of individual cells into organized tissues.

Non-selective junctional communication between some different mammalian cell types in primary culture.

Intercellular transfer of tritium-labelled uridine nucleotides has been used to detect junctional communication between various cell types in primary culture. Epidermal keratinocytes, melanocytes and dermal fibroblasts from new-born-mouse skin, and epithelial cells from baby mouse kidney form communicating junctions in all possible homologous and heterologous combinations. This lack of detectable communication specificity between cells in primary culture contrasts with the specificity shown by some established cell lines.

Communication compartments in mixed cell cultures.

Mixed cultures of epithelial (BRL) cells and fibroblasts (BHK), which sort themselves out into separate domains of each cell type, form communication compartments. Electrical coupling, dye coupling and metabolic coupling measurements have been used to show that small ions and molecules can move freely via intercellular junctions between all the cells in a domain, while their movement across the boundaries between domains is severely restricted. Metabolic coupling is the most sensitive method for detecting trans-boundary communication but the results obtained from all three methods are compatible. The data suggest the reduced transfer across the boundaries is due to fewer channels, resulting from a lower frequency of junction formation between heterologous cells, rather than to channels of smaller diameter. Concentration gradients of small cytoplasmic molecules can be established within these communication compartments which are similar to those predicted to explain pattern formation in developing systems. It is suggested that the cell surface features which cause this sorting out are also responsible for the reduced frequency of heterologous junction formation and hence for compartmentalization.

Mechanism of inhibition of junctional communication between animal cells by phorbol ester.

The tumour promotor 4 beta-phorbol 12-myristate 13-acetate (TPA) reduces the rate of junction formation between V79 Chinese hamster lung cells in culture but not between BHK21/13 Syrian hamster fibroblasts. TPA may act on all cells but only affect junction formation in those situations where the rate of junction formation is already low.