Vsx1, a rapidly evolving paired-like homeobox gene expressed in cone bipolar cells.

The paired-like homeodomain (HD) protein Chx10 is distinguished by the presence of the CVC domain, a conserved 56 amino acid sequence C-terminal to the HD. In mammals, Chx10 is essential both for the proliferation of retinal progenitor cells and for the formation or survival of retinal bipolar interneurons. We describe the cloning and characterization of a mouse Chx10 homologue, Vsx1; phylogenetic analysis suggests that Vsx1 and its putative vertebrate orthologues have evolved rapidly. Vsx1 expression in the adult is predominantly retinal. Whereas Chx10 is expressed both in retinal progenitors in the developing eye and apparently in all bipolar cells of the mature retina, Vsx1 expression is first detected in the eye at postnatal day 5, where it is restricted to cone bipolar cells.

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis.

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.

PHR1 encodes an abundant, pleckstrin homology domain-containing integral membrane protein in the photoreceptor outer segments.

We cloned human and murine cDNAs of a gene (designated PHR1), expressed preferentially in retina and brain. In both species, PHR1 utilizes two promoters and alternative splicing to produce four PHR1 transcripts, encoding isoforms of 243, 224, 208, and 189 amino acids, each with a pleckstrin homology domain at their N terminus and a transmembrane domain at their C terminus. Transcript 1 originates from a 5'-photoreceptor-specific promoter with at least three Crx elements ((C/T)TAATCC). Transcript 2 originates from the same promoter but lacks exon 7, which encodes 35 amino acids immediately C-terminal to the pleckstrin homology domain. Transcripts 3 and 4 originate from an internal promoter in intron 2 and either include or lack exon 7, respectively. In situ hybridization shows that PHR1 is highly expressed in photoreceptors, with lower expression in retinal ganglion cells. Immunohistochemistry localizes the PHR1 protein to photoreceptor outer segments where chemical extraction studies confirm it is an integral membrane protein. Using a series of PHR1 glutathione S-transferase fusion proteins to perform in vitro binding assays, we found PHR1 binds transducin betagamma subunits but not inositol phosphates. This activity and subcellular location suggests that PHR1 may function as a previously unrecognized modulator of the phototransduction pathway.

Spatiotemporal distribution of SPARC/osteonectin in developing and mature chicken retina.

Expression of SPARC (Secreted Protein, Acidic, Rich in Cysteine), a counteradhesive, calcium-binding extracellular matrix (ECM) glycoprotein, is associated with several morphogenetic events during early development. In this study, changes in the spatiotemporal distribution of SPARC transcripts and the protein during chicken retinal development were documented by in situ hybridization and indirect immunofluorescence microscopy. SPARC transcripts were first detected within the proliferating neural ectoderm at embryonic day 4. 5 (E4.5), followed short thereafter (E5) by appearance of SPARC. SPARC was enriched within the inner plexiform layer (IPL) by E10 and within the outer plexiform layer (OPL) by E14, several days after these layers became morphologically distinct. Significant levels of SPARC transcripts were first observed within the ganglion cell layer (GCL) at E17 prior to accumulation of SPARC within the nerve fiber layer, seen first at E20. SPARC protein was first detected within the developing retinal pigment epithelium (RPE) at E10 and increased significantly at RPE cells ceased to proliferate and continued differentiating. Of special note was the restriction of SPARC to the basal-half of the RPE cells. SPARC transcripts were similarly distributed in the adult retina, but at lower levels than in the period just prior to hatching. In the adult retina SPARC was retained in the nerve fiber layer and present in the inner nuclear layer (INL) and outer nuclear layer (ONL), but lost from the IPL and OPL. These changes in expression pattern with time indicate that SPARC is developmentally regulated and therefore may have important function(s) in both morphological development of the retina and functioning of the mature eye.

Ocular retardation mouse caused by Chx10 homeobox null allele: impaired retinal progenitor proliferation and bipolar cell differentiation.

Ocular retardation (or) is a murine eye mutation causing microphthalmia, a thin hypocellular retina and optic nerve aplasia. Here we show that mice carrying the OrJ allele have a premature stop codon in the homeobox of the Chx10 gene, a gene expressed at high levels in uncommitted retinal progenitor cells and mature bipolar cells. No CHX10 protein was detectable in the retinal neuroepithelium of orJ homozygotes. The loss of CHX10 leads both to reduced proliferation of retinal progenitors and to a specific absence of differentiated bipolar cells. Other major retinal cell types were present and correctly positioned in the mutant retina, although rod outer segments were short and retinal lamination was incomplete. These results indicate that Chx10 is an essential component in the network of genes required for the development of the mammalian eye, with profound effects on retinal progenitor proliferation and bipolar cell specification or differentiation. off

Developmental expression of a novel murine homeobox gene (Chx10): evidence for roles in determination of the neuroretina and inner nuclear layer.

Few potential regulatory proteins of vertebrate retinal development have been identified. We describe a 39 kDa murine polypeptide (Chx10) with a homeodomain 82% identical to that of the nematode protein ceh-10. In the developing mouse, the Chx10 transcript is expressed throughout the anterior optic vesicle and all neuroblasts of the optic cup. In the mature retina, the Chx10 protein is restricted to the inner nuclear layer, in which its expression decreases from the outer to the inner margin. Chx10 transcripts are also detected in regions of the developing thalamus, hindbrain, and ventral spinal cord. The data suggest that Chx10 plays critical roles in the formation of the neuroretina and in the development and maintenance of the inner nuclear layer.

Sustained increases in cytosolic calcium during T lymphocyte allosensitization, proliferation, and acquisition of locomotor function.

Activation of resting T cells is accompanied by an increase in cytosolic calcium ([Ca2+]i). However, the role of [Ca2+]i in the effector function of allosensitized cells, and how this may affect the evolution of the allograft response is unknown. To evaluate this more directly, we determined [Ca2+]i in both unsensitized T cells (C57BL/6 murine thymocytes) and in allosensitized T cells derived from different days of a C57BL/6 anti-DBA/2J mixed leukocyte culture. To correlate potential changes in [Ca2+]i with concomitant development of T cell effector function, the [Ca2+]i, proliferation (3HTdR uptake), and random locomotion (in vitro modified Boyden chamber assay) of the same cells was assayed simultaneously. Allosensitized T cells exhibited higher (P less than 0.05) [Ca2+]i than unsensitized thymocytes on all days of culture tested. Further, there was a progressive rise in [Ca2+]i during the course of allosensitization. Con A stimulated an increase in [Ca2+]i over basal levels (P less than .05) for all cell types. A rise in [Ca2+]i preceded the onset of maximal allosensitized T cell proliferation (which peaked at day 7) and this continued to increase even after completion of DNA synthesis. In contrast, optimal T cell locomotion coincided with maximal [Ca2+]i, well after cell division had occurred. Prostaglandin E2, a known inhibitor of lymphocyte function, did not alter either basal or Con A-stimulated [Ca2+]i in thymocytes or MLC cells. These results indicate that [Ca2+]i signaling persists long after initial T lymphocyte alloactivation, and is maintained during DNA synthesis and acquisition of locomotor capacity. Furthermore, the inhibitory effects of PGE2 on allosensitized T lymphocyte function may be mediated by a calcium-independent mechanism.

Antineoplastic drug cytotoxicity in a human bladder cancer cell line: implications for intravesical chemotherapy.

The clonogenic survival of MGH-U1 human bladder carcinoma cells treated with melphalan, cisplatin, mitomycin-C, adriamycin, vincristine and 5-fluorouracil was measured to determine the relative contribution of drug concentration and duration of exposure to cytotoxicity and to measure the relative cytotoxic effects of these agents used in intravesical chemotherapy. The survival curves were plotted as a function of log (C X T) and were fitted using a linear least squares analysis. The survival was the same for any given C X T whether this was determined by varying concentration or by varying the duration of exposure in the cases of melphalan, cisplatin, adriamycin, mitomycin-C and 5-fluorouracil. However, duration of exposure was more important than was drug concentration in the case of vincristine cytotoxicity. By utilizing the slope of the log (survival fraction) as a function of log (C X T), the relative cytotoxicity of each agent was determined. Mitomycin C, melphalan, adriamycin and cisplatin had comparable activity in this cell line, whereas vincristine and 5-fluorouracil demonstrated much lower cytotoxicity. We conclude that: mitomycin-C, adriamycin and melphalan were the agents with the greatest cytotoxic efficacy; determination of survival as a function of C X T can be used to separate the relative importance of concentration and of duration of exposure. the cytotoxicity of 5/6 drugs studied was equal when the C X T was kept constant but concentration and exposure times were varied.

Cytotoxicity of cisplatin and cisdiammine-1,1-cyclobutane dicarboxylate in MGH-U1 cells grown as monolayers, spheroids, and xenografts.

The cytotoxicity of cisplatin and cisdiammine-1,1-cyclobutane dicarboxylate (CBDCA) was examined using the MGH-U1 human bladder carcinoma cell line, grown as monolayer cultures, multicellular tumor spheroid(s) (MTS), and xenografts in immune-deprived CBA/CaJ mice. The cell survival of exponentially growing monolayers and MTS treated with cisplatin declined in a monoexponential fashion with a concentration of drug resulting in 10% colony survival (D10) of 7.75 micrograms/ml and 9.5 micrograms/ml, respectively. MTS growth delay determination demonstrated a drug concentration-dependent increase in growth delay and a correlation between decreasing surviving fraction and increasing growth delay. In vivo treatment of MGH-U1 xenografts with cisplatin caused a modest decrease in surviving fraction although the xenografted cells treated in vitro demonstrated the same sensitivity to cisplatin as those cells maintained continuously in vitro. The D10 for CBDCA treatment was 246 micrograms/ml for exponentially growing monolayer cells and 196 micrograms/ml for MTS. Growth delay studies with CBDCA showed a concentration-dependent increase in spheroid growth delay and a correlation between decreasing surviving fraction and growth delay similar to cisplatin. The conclusions were that: 1) cisplatin and CBDCA did not have any difficulty penetrating into spheroids, 2) both agents appeared to be active against the noncycling poorly nourished cells found near the necrotic center of spheroids, 3) both cisplatin and CBDCA were cytotoxic toward MGH-U1 cells but cisplatin was 20-30 times more effective, and 4) the limited cytotoxic effect of cisplatin in vivo may be due to the low area under the concentration times the time curve achieved in vivo and not due to intrinsic cell resistance.

Cytotoxicity of adriamycin in MGH-U1 cells grown as monolayer cultures, spheroids, and xenografts in immune-deprived mice.

The cytotoxic activity of Adriamycin was examined in the MGH-U1 human bladder carcinoma line, grown as monolayer culture, as spheroids, and as xenografts in immune-deprived mice. The MGH-U1 cells grown as spheroids were much more resistant to Adriamycin (concentration of drug resulting in 37% cell survival, 4.5 micrograms/ml) than when treated as monolayer cultures (concentration of drug resulting in 37% cell survival, 0.9 microgram/ml). Adriamycin fluorescence was demonstrated only in the outer two layers of cells forming the spheroids, suggesting that limited drug penetration is an important factor in the resistance of spheroids to Adriamycin. Sequential trypsinization of spheroids 750 micron in diameter allowed us to determine the cytotoxic effects of Adriamycin in MGH-U1 cells derived from different depths of the spheroid. We found that cells near the surface of the spheroid had a survival similar to those of exponentially growing monolayer cells treated with Adriamycin. Cells located in the middle of the viable rim were more resistant to Adriamycin, and those found near the necrotic center were most resistant to Adriamycin. The effects of Adriamycin treatment on spheroid growth delay were determined also. In spite of a small cytotoxic effect on the clonogenic fraction of cells in MGH-U1 spheroids, the growth delay effect of Adriamycin in intact spheroids was marked. This observation is consistent with Adriamycin killing primarily the cells in the outer layers of the spheroid, where most of the proliferation in the spheroid occurs. In vivo treatment of MGH-U1 xenografts with Adriamycin followed by assessment of cell survival in vitro showed minimal evidence of cytotoxicity, consistent with the poor drug penetration observed in the spheroid model. These studies suggest that: (a) Adriamycin penetrates poorly into solid tissues; (b) in vitro clonogenic survival following Adriamycin exposure of a cell suspension may predict falsely for drug sensitivity to chemotherapy; (c) a small decrease in clonogenic survival can be translated into a long growth delay but, ultimately, the tumor regrows because some clonogenic cells are spared; and (d) for Adriamycin, the spheroid model more closely parallels the in vivo effects than does monolayer culture. The use of the spheroid model for the study of Adriamycin cytotoxicity gives further insight into the action of this drug in solid tumors.

Antitumor activity of N-phosphonacetyl-L-aspartic acid in combination with nitrobenzylthioinosine.

N-Phosphonacetyl-L-aspartic acid (PALA) resistance may be due to the ability of tumor cells to utilize preformed circulating pyrimidine nucleosides, thereby overcoming the block of de novo pyrimidine biosynthesis which PALA causes. To test this hypothesis we examined the effects of PALA and nitrobenzylthioinosine (NBMPR) alone and in combination on B16 melanoma cells in vitro using a clonogenic assay and in vivo using growth delay. In medium containing purine and pyrimidine nucleosides at a final concentration of 28 microM, exposure to PALA (100 microM) alone or to NBMPR (10 microM) alone for periods up to 72 hr did not result in any cytotoxicity. However, exposures to PALA (100 microM) plus NBMPR (10 microM) resulted in a decrease in clonogenic survival to 0.011 at 72 hr. In medium without nucleosides, PALA (100 microM) exposure for 72 hr caused a similar decrease in survival to 0.015, whereas NBMPR (10 microM) had no effect on survival. The addition of uridine resulted in a concentration-dependent reversal of the cytotoxic effects of PALA. C57 Bl female mice bearing B16 melanoma were treated intraperitoneally daily for 4 days with PALA, the phosphate of NBMPR (NBMPR-P), or PALA plus NBMPR-P. PALA, 300 mg/kg daily X 4, resulted in a 6-day tumor growth delay but NBMPR-P, 100 mg/kg daily X 4, had no effect. PALA, 150 mg/kg daily X 4, plus NBMPR, 50 or 100 mg/kg daily X 4, resulted in a 6-day tumor growth delay also. These studies demonstrate that: (1) circulating pyrimidine nucleosides are determinants of the cytotoxic effects of PALA; (2) in vitro PALA and NBMPR combine to cause significant cytotoxicity whereas either agent alone has no effect; (3) in vivo the combination of PALA and NBMPR-P results in the same antitumor affect as PALA alone at twice the dose; and (4) due to an increase in animal toxicity, no therapeutic advantage could be demonstrated for the combination over PALA alone in vivo. We conclude that the cytotoxic effect of PALA is modulated by the levels of the preformed circulating nucleosides and that combining PALA with an inhibitor of salvage pyrimidine uptake would not increase the therapeutic efficacy of PALA because of an increase in toxicity.