Identification of factors important for the success of suicide gene therapy after a comparative study of Varicella zoster and Herpes simplex viral thymidine kinases efficacy on breast cancer cells.

One of the most frequently studied therapeutic strategies in the field of suicide gene therapy is based on the expression by tumor cells of the Herpes simplex virus thymidine kinase gene (HSVtk) followed by a ganciclovir (GCV) treatment. In order to investigate the potential of other enzyme/prodrug strategies, we studied in vitro and in vivo the ability of the Varicella zoster virus thymidine kinase gene (VZVtk) to act as a suicide gene and to kill non-transduced bystander cells, and compared this activity to that of its HSV counterpart. Four different antiviral compounds were tested as prodrugs. Our comparative study demonstrates the superiority of the HSVtk/GCV system among the different combinations tested and underlines the importance of both the tumor cell type and the prodrug in the success of a prodrug/suicide gene strategy.

Down-regulation of vascular endothelial growth factor by tissue inhibitor of metalloproteinase-2: effect on in vivo mammary tumor growth and angiogenesis.

The tissue inhibitor of metalloproteinases-2 (TIMP-2) has at least two independent functions, i.e., regulation of matrix metalloproteinases and growth promoting activity. We investigated the effects of TIMP-2 overexpression, induced by retroviral mediated gene transfer, on the in vivo development of mammary tumors in syngeneic mice inoculated with EF43.fgf-4 cells. The EF43.fgf-4 cells established by stably infecting the normal mouse mammary EF43 cells with a retroviral expression vector for the fgf-4 oncogene, are highly tumorigenic and overproduce vascular endothelial growth factor (VEGF). Despite a promotion of the in vitro growth rate of EF43.fgf-4 cells overexpressing timp-2, the in vivo tumor growth was delayed. At day 17 post-cell injection, the volume of tumor derived from TIMP-2-overexpressing cells was reduced by 80% as compared with that obtained with control cells. Overexpression of TIMP-2 was associated with a down-regulation of VEGF expression in vitro and in vivo, a reduction of vessel size, density, and blood supply in the induced tumors. In addition, TIMP-2 completely inhibited the angiogenic activity of EF43.fgf-4 cell-conditioned medium in vitro using a rat aortic ring model. Our findings suggest that overexpression of TIMP-2 delays growth and angiogenesis of mammary carcinoma in vivo and that down-regulation of VEGF expression may play an important role in this TIMP-2-mediated antitumoral and antiangiogenic effects. Finally the in vivo delivery of TIMP-2, as assessed by i.v. injection of recombinant adenoviruses vectors, significantly reduced the growth of the EF43.fgf-4-induced tumors. This effect of TIMP-2 was shown to be equally comparable with that of angiostatin, a known potent inhibitor of angiogenesis.

The role of cellular- and prodrug-associated factors in the bystander effect induced by the Varicella zoster and Herpes simplex viral thymidine kinases in suicide gene therapy.

To investigate the factors influencing the bystander effect--a key element in the efficacy of suicide gene therapy against cancer--we compared the effect triggered by four extremely efficient gene/prodrug combinations, i.e., VZVtk/BVDU, the thymidine kinase of Varicella zoster virus associated with (E)-5-(2-bromovinyl)-2'-deoxyuridine; VZVtk/BVaraU, the same enzyme associated with (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil; HSVtk/BVDU, the association of the Herpes simplex virus thymidine kinase with BVDU; and the classical HSVtk/GCV (ganciclovir) paradigm. The cells used, the human MDA-MB-435 breast cancer, and the rat 9L glioblastoma lines were equally sensitive in vitro to these four associations. In both cell types, the combinations involving pyrimidine analogues (BVDU, BVaraU) displayed a smaller bystander killing than the combination involving the purine analogue (GCV). In addition, the bystander effect induced by all the tk/prodrug systems was reduced in MDA-MB-435 cells in comparison to 9L cells; albeit, the viral kinases were produced at a higher level in the breast cancer cells. All systems induced apoptotic death in the two cell types, but the MDA-MB-435 cells, deprived of connexin 43, were noncommunicating in striking contrast with the 9L cells. That functional gap junctions have to be increased in order to improve the breast cancer cell response to suicide gene therapy was demonstrated by transducing the Cx43 gene: this modification enhanced the bystander effect associated in vitro with GCV treatment and, by itself, decreased the tumorigenicity of the untreated cells. However, the noncommunicating MDA-MB-435 cells triggered a significant bystander effect both in vitro and in vivo with the HSVtk/GCV system, showing that communication through gap junctions is not the only mechanism involved.

Progression in MCF-7 breast cancer cell tumorigenicity: compared effect of FGF-3 and FGF-4.

The transforming properties of fibroblast growth factor 3 (FGF-3) were investigated in MCF7 breast cancer cells and compared to those of FGF-4, a known oncogenic product. The short form of fgf-3 and the fgf-4 sequences were each introduced with retroviral vectors and the proteins were only detected in the cytoplasm of the infected cells, as expected. In vitro, cells producing FGF-3 (MCF7.fgf-3) and FGF-4 (MCF7.fgf-4) displayed an amount of estrogen receptors decreased to around 45% of the control value. However, MCF7.fgf-3 cell proliferation remained responsive to estradiol supply. The sensitivity of the MCF7.fgf-4 cells, if existant, was masked by the important mitogenic action exerted by FGF-4. In vivo, the MCF7.fgf-3 and MCF7.fgf-4 cells gave rise to tumors under conditions in which the control cells were not tumorigenic. Supplementing the mice with estrogen had the paradoxical effect of totally suppressing the start of the FGF-3 as well as the FGF-4 tumors. Tumorigenicity in the presence of matrigel was similar for MCF7.fgf-3 and control cells and was increased by estrogen supplementation. Once started, the MCF7.fgf-4 tumors grew with a characteristic high rate. Remarkably, FGF-4 but not FGF-3, stimulated the secretion of vascular endothelial growth factor (VEGF165) without altering the steady-state level of its mRNA, suggesting a possible regulation of VEGF synthesis at the translational level in MCF7 cells. The increased VEGF secretion is probably involved in the more aggressive phenotype of the MCF7.fgf-4 cells while a decreased dependence upon micro-environmental factors might be part of the increased tumorigenic potential of the MCF7.fgf-3 cells.

Comparative in vitro and in vivo cytotoxic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative, (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU), against tumor cells expressing either the Varicella zoster or the Herpes simplex virus thymidine kinase.

The inhibitory effects of (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and its arabinosyl derivative (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil (BVaraU) on the growth of both MDA-MB-435 human breast carcinoma and 9L rat gliosarcoma cells expressing the thymidine kinase (tk)-encoding gene of the Varicella zoster virus (VZV) or the Herpes simplex virus (HSV) were evaluated. In vitro, BVDU and BVaraU effectively killed both cell types expressing VZVtk, with 50% inhibitory concentration values ranging from 0.06 to 0.4 microM, whereas ganciclovir (GCV) lacked activity. On HSVtk+ cells, BVDU had high cytotoxic activity, with 50% inhibitory concentration values that were similar to those of GCV, whereas BVaraU was inactive. In vivo, BVDU applied intraperitoneally caused a 50% tumor growth inhibition in nude mice inoculated subcutaneously with VZVtk+ as well as HSVtk+ mammary tumor cells. In mice and at variance with the in vitro results, BVaraU had very little activity against the VZVtk+ mammary cells; GCV had the highest activity on the HSVtk+ cells, resulting in a 50% eradication of the tumors. With the 9L rat gliosarcoma model, the VZVtk/BVDU system completely failed to inhibit the development of VZVtk+ glioma tumors induced subcutaneously in syngeneic rats, although BVDU had a similar 45-minute half-life in both rats and mice. Factors other than degradation of the prodrug and related to the mode of action of these analogs are possibly involved in the observed discrepancies between the in vitro and in vivo results.

Demonstration in vivo that stromelysin-3 functions through its proteolytic activity.

Stromelysin-3 (ST3), a matrix metalloproteinase (MMP) expressed in aggressive carcinomas, has been shown to promote tumor development in different in vivo experimental models. However, the inability of its mature form to degrade extracellular matrix components casts doubt on whether ST3 functions in vivo as a protease. In this study, we evaluated whether the ST3 tumor-promoting effect could be ascribed to its proteolytic activity and whether this putative protease could be targeted with MMP inhibitors. Catalytically inactive mutant cDNA of human (h) ST3 or mouse (m) ST3 were generated and transfected into MCF7 cells. When injected into nude mice in the presence of matrigel, the mutant-bearing cells did not exhibit the enhanced tumorigenicity elicited by MCF7 cells transfected with wild-type ST3 cDNA. In a second approach, TIMP2 overproduction in MCF7 cells expressing hST3 was induced by retroviral infection. The co-expression of ST3 and TIMP2 failed to enhance the tumorigenicity of MCF7 cells. Notably, matrigel depleted of low-molecular-weight proteins and growth factors failed to promote the tumorigenicity of ST3-expressing MCF7 cells. These findings provide the first in vivo evidence that ST3 is indeed a protease that can modulate cancer progression by remodeling extracellular matrix and probably by inducing it to release the necessary microenvironmental factors. Thus, ST3 represents an interesting target for specific MMP inhibition.

FGF-3 and FGF-4 elicit distinct oncogenic properties in mouse mammary myoepithelial cells.

Fibroblast Growth Factors 3 (FGF-3) and 4 (FGF-4) were compared for the effects they each exert on EF43 mouse cells. This non-transformed mammary cell line appears to be myoepithelial mainly because it expresses alpha-smooth muscle actin. The EF43 cells were infected with similar vectors that carry either the short fgf-3 sequence (the product of which goes into the secretory pathway), fgf-4 or the selection gene only as control. In syngeneic animals, EF43.fgf-3 cells were tumorigenic only when orthotopically implanted whereas EF43.fgf-4 cells invariably gave rise to aggressive tumors. However, both tumor types were metastatic as evidenced by the blue micrometastases observed when the implanted cells expressed lacZ. In vitro, the FGF-3 producing cells were strongly invasive in matrigel coated chambers whereas the EF43.fgf-4 cells only were invasive in type I-collagen gels. Interestingly, FGF-3 production greatly stimulated the synthesis of pro-MMP-9 (Matrix Metalloprotease-9) and, to a lesser extent, that of pro-MMP-2. FGF-3 also up-regulated the production of plasminogen activators. In contrast, FGF-4 had no effect on these secretions and the medium conditioned by the EF43.fgf-4 cells displayed the largest plasminogen activator-inhibitor activity. These results show that FGF-3 and FGF-4 have distinct mechanisms of action on myoepithelial cells.

Inhibition of stromal matrix metalloproteases: effects on breast-tumor promotion by fibroblasts.

Co-injection of fibroblasts with human epithelial breast-tumor MCF7 cells in the presence of Matrigel enhances tumor growth in nude mice. While most of the matrix metalloproteinases (MMPs) have been shown to be produced by stromal cells, tumor cells such as MCF7 cells are unable to produce MMPs. We therefore, hypothesized that the tumor-promoting effect of fibroblasts could be related to their production of MMPs. In order to inhibit stromal proteases, over-production of TIMP-2 was induced in MCF7 cells by in vitro retroviral-mediated gene transfer. TIMP-2-producing MCF7 cells were then co-injected with fibroblasts into nude mice. Alternatively, we evaluated the effect of Batimastat, a synthetic inhibitor of MMPs, on the tumorigenicity of MCF7 cells co-inoculated with fibroblasts into nude mice. Both physiological (TIMP-2) and synthetic (Batimastat) inhibitors of MMPs were able to abolish the tumor-promoting effect of fibroblasts. On the contrary, they failed to modulate the tumorigenicity of MCF7 cells injected alone. Interestingly, Matrigel from which low-molecular-weight proteins or growth factors had been removed failed to favor the tumorigenicity of MCF7 cells inoculated with fibroblasts. These findings emphasize the importance of fibroblasts in cancer progression, and suggest that their role could be related at least in part to production of proteases which can induce the release of factors from the extracellular matrix.

Potential of Varicella zoster virus thymidine kinase as a suicide gene in breast cancer cells.

To investigate the potential of the thymidine kinase gene from Varicella zoster virus (VZVtk) to act as a suicide gene, VZVtk was transferred via a dicistronic retroviral construct into MCF7, T-47D and MDA-MB-435 human breast cancer cells. The cytotoxicity of antiviral drugs was then evaluated in vitro on the wild-type and transduced cells. Acyclovir and ganciclovir did not show any selective toxicity for the modified cells. In contrast, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was extremely toxic for the VZVtk expressing cells, with IC50 values of 0.6 microM, 0.1 microM and 0.06 microM for MCF7, T-47D and MDA-MB-435 cells, respectively. The selectivity index of BVDU (ie the IC50 value ratio of the wild-type to the VZVtk cells) was 400 for MCF7, 750 for T-47D and 2000 for MDA-MB-435 cells. To test the system in vivo, VZVtk carrying MDA-MB-435 cells were inoculated subcutaneously into nude mice. An intraperitoneal treatment with BVDU administered at the emergence of the tumors, led to a prolonged arrest of the tumor growth and a reduced tumor mass. This effect was BVDU dose-dependent. No bystander effect of BVDU killing could be demonstrated in vitro on mixed populations of VZVtk positive and negative MDA-MB-435 cells. However, an important bystander effect was observed in identical experiments performed on 9L rat gliosarcoma cells infected with the VZVtk-carrying vector. These results demonstrate the efficiency of VZVtk as a suicide gene when BVDU is used as prodrug. The bystander effect measured in vitro, depends however on the tumoral cell type used.

Angiogenesis by fibroblast growth factor 4 is mediated through an autocrine up-regulation of vascular endothelial growth factor expression.

The infection of normal mouse mammary EF43 cells by a retroviral vector carrying either Fgf-3 (EF43.Fgf-3) or Fgf-4 (EF43.Fgf-4) cDNA resulted in the transformation of cells displaying different tumorigenic potentials in nude mice (A. Hajitou and C-M. Calberg-Bacq, Int. J. Cancer, 63: 702-709, 1995). EF43.Fgf-4 produced rapidly developing tumors at all sites of inoculation, whereas EF43.Fgf-3 produced slowly growing tumors only in the mammary fat pad. Cells infected with the vector carrying the selection gene alone (EF43.C) were not tumorigenic. The angiogenic properties of these cells were tested in an in vitro angiogenesis model using human umbilical vein endothelial cells (HUVECs) cultured at the surface of a type I collagen gel and their capacity to form tube-like structures on invasion of the gel. Only the conditioned medium (CM) of EF43.Fgf-4 induced an angiogenic morphotype in HUVECs. In parallel, the mRNA expression of matrix metalloproteinase 1 and c-ETS-1 was increased in the HUVECs displaying a differentiated phenotype, whereas the tissue inhibitor of matrix metalloproteinase 1 mRNA level was decreased. Recombinant human fibroblast growth factor 4 (FGF-4) did not induce an angiogenic phenotype in HUVECs by itself. By Western blot analysis, a high expression of vascular endothelial growth factor (VEGF) was detected in the EF43.Fgf-4 CM. This result was confirmed by Northern blot analysis of total RNA extracted from the three cell types; the steady-state level of VEGF mRNA was low and equivalent in EF43.C and EF43.Fgf-3, whereas it was strongly increased in EF43.Fgf-4. Culturing EF43 cells carrying only the selection gene with increasing concentrations of recombinant human FGF-4 resulted in a dose-dependent stimulation of VEGF. The induction of the angiogenic morphotype and the parallel modulations of the biosynthetic phenotype in HUVECs were completely suppressed by adding a neutralizing antibody directed against VEGF to EF43.Fgf-4 CM. Furthermore, inhibition of protein kinase C by bisindoylmaleimide suppressed the angiogenic phenotype induced by the CM of EF43.Fgf-4. Our results point to an indirect angiogenic activity of FGF-4 through the autocrine induction of VEGF secretion by EF43.Fgf-4 cells, an original signaling pathway that might be significant in tumor progression and metastasis.

Fibroblast growth factor 3 is tumorigenic for mouse mammary cells orthotopically implanted in nude mice.

Fibroblast growth factor-3 (Fgf-3) is involved in mouse mammary tumorigenesis since the Fgf-3 gene is a main target for mouse mammary tumor virus (MMTV) insertional activation. Its action has been correlated with the appearance of pregnancy-dependent tumors. We describe here the effects on normal mouse mammary EF43 cells of the short Fgf-3 protein form which enters the secretory pathway. The genes, Fgf-3 AUG or Fgf-4 for comparison, were introduced in the mammary cells by means of retroviral vectors. Fgf-3 expression did not modify EF43 cell morphology, had no effect on growth in soft agar nor on the inhibitory action exerted on cell growth by TGF-beta 1; however, it allowed the cells to grow under low serum conditions in the absence of insulin and EGF. The Fgf-3-expressing cells were not tumorigenic in nude mice when injected s.c., but tumors developed when the cells were implanted in the mammary gland. The tumors appeared after some latency; they had a slow growth phase followed by a phase of increased growth rate. An identical tumoral growth pattern was observed in ovariectomized nude mice. These results show that the secreted Fgf-3 form can initiate tumorigenesis and that the induced tumors are hormone-independent. The mammary-gland environment, however, is required for the EF43 cells to grow and differentiate. During that process, which resembles natural cell growth during mammary-gland development at pregnancy, the cells could pass through a stage which is specifically sensitive to Fgf-3.

Mouse mammary-tumor virus activates Fgf-3/Int-2 less frequently in tumors from virgin than from parous mice.

Tumorigenesis by mouse mammary-tumor virus (MMTV) involves proviral disruption and transcriptional activation of a number of cellular oncogenes, generically termed Int. The frequencies with which different Int genes are activated in different mouse strains can be quite variable, and previous surveys have suggested that insertions at Int-2/Fgf-3 occur primarily in strains that develop pregnancy-dependent mammary tumors. To address this issue, we have determined the relative contributions of 5 known Int genes (Wnt-1, Wnt-3, Fgf-3, Fgf-4 and Int-3) in mammary tumors from virgin BR6 and multiparous BR6, BALB/cfBR6 and RIII mice. Whereas Fgf-3 was implicated in 66%, 80% and 92% of the tumors from the respective parous animals, only 20% of the tumors from virgin mice expressed Fgf-3. This reduced involvement of Fgf-3 was compensated by proviral insertions in Fgf-4, Int-3 and Wnt-3, but the frequency of Wnt-1 activation was relatively constant. These data strengthen the link between Fgf-3 and a pregnancy-dependent phenotype and suggest that, in the strains analyzed, the frequency of Int-gene activation was influenced more by the hormonal status than by the genetic background.

Modulation of collagen and fibronectin synthesis in fibroblasts by normal and malignant cells.

The influence of various normal and malignant human cells on the level of collagen synthesis by human fibroblasts was tested in coculture. As revealed by immunoperoxidase staining, in cocultures with breast adenocarcinoma cells (MCF7, SA52, T47D) fibroblasts synthesized collagen while tumor cells did not. Fibroblasts displayed increased collagen production without change in the overall protein synthesis. Several other types of cells derived from normal human tissues (keratinocytes, normal mammary cells) or from fibrosarcoma, melanoma, cervical carcinoma, choriocarcinoma, or other breast adenocarcinoma (SW613, MDA, BT20) did not affect collagen synthesis of fibroblasts. Although to a lesser extent, this stimulating effect was reproduced by using the conditioned medium (CM) of the active cells but not with CM of the other cell types. A slight stimulation was also obtained when tumoral MCF7 cells and fibroblasts shared the same medium but were physically separated, suggesting that close contact was required for optimal stimulation of collagen synthesis. The collagen synthesis stimulating activity was not related to a modification of fibroblast proliferation rate. The production of collagen types I, III, and VI and fibronectin were increased in cocultures of fibroblasts with MCF7 cells. The increased synthesis of collagen types I and III and fibronectin was paralleled by similar changes in the steady-state level of their mRNAs. On the contrary, the increased production of collagen type VI appeared regulated at a post-transcriptional level.

Mouse mammary tumor virus (MMTV) infection in SWISS and RIII mice. Correlation between resistance to exogenous infection and anti-MMTV serum response.

Host-virus relationships were examined in mice from the two mouse mammary tumor virus (MMTV)-infected strains SWISS MB+ and RIII, which harbour the same MMTV variant, and from the derived sublines Swiss MB- and RIIIf, which were freed of milk-borne MMTV by foster-nursing. These two strains are not phylogenetically related, the SWISS strain bearing the endogenous Mtv-3 locus in its DNA. In RIII and SWISS MB+ mice, the incidence of early mammary tumors, which was of 96% and 8%, respectively, was correlated to the level of MMTV expression in milk. In the SWISS MB-line, a non-coordinate expression of the provirus associated with the Mtv-3 locus was observed in the mammary glands, the salivary glands and the spleen. This expression was not tumorigenic and was characterized by the presence of the p28 gag antigen and the absence of the gp52 env antigen, except, however, in mammary glands of elder mice where traces of gp52 were found. In the mammary glands of SWISS MB+ mice, the expression of the Mtv-3 locus was masked by large amounts of antigens resulting from exogenous virus expression. RIIIf mice were MMTV-negative. Viral antigens coexisted with anti-MMTV antibodies in the serum of infected and tumor-bearing mice, but not in the form of immune complexes as verified by a method that allowed to detect specific antigen-containing-soluble immune complexes. An anti-MMTV serum reactivity was also detected in SWISS MB- and RIIIf mice. However, the serum response was higher in the two SWISS lines than in the two RIII lines. Except in tumor-bearing mice, the anti-MMTV response was not significantly modified by the presence of exogenous virus and thus resulted essentially from exposure to endogenous MMTV expression. In experimental infection studies, RIII mice were more susceptible to MMTV infection than SWISS mice. The correlation between resistance to MMTV infection and serum response to endogenous MMTV expression, suggests that the non-tumorigenic expression of an endogenous provirus can protect at least partially, against exogenous MMTV infection.

Production of mouse mammary tumor virus upon transfection of a recombinant proviral DNA into cultured cells.

We have investigated the intracellular proteins synthesized in rat XC and feline kidney cells transfected with endogenous mouse mammary tumor virus (MMTV) proviral DNA. The endogenous provirus GR40, associated with the Mtv-8 locus, directs the synthesis of gag proteins indistinguishable from those found in MMTV-infected cells. The env precursor Pr73env and the mature gp52 proteins could not be detected in these cells. Instead an env-related protein of 68K is synthesized. In contrast to this endogenous provirus, a cloned exogenous proviral variant directs the synthesis of apparently normal env proteins upon transfection into the same cell lines. These results suggest that the env gene of the endogenous MMTV provirus GR40 is defective. The exogenous proviral variant is not expected to synthesize virus particles since it carries a rearrangement in the gag gene. In order to obtain an MMTV provirus capable of correctly expressing both gag and env functions, we have constructed a hybrid endogenous-exogenous provirus containing the 5' long terminal repeat (LTR)-gag of GR40 and the pol-env-3' LTR of the exogenous provirus. Upon transfection into feline kidney cells, this hybrid provirus directed the synthesis of apparently authentic gag and env proteins. Further, virus particles can be detected in the culture medium of the transfected cells by electron microscopy. Viral proteins obtained from viral particles banded in a sucrose gradient were detected by immunoprecipitation.

Natural infection of Swiss mice with mouse mammary tumor virus (MMTV): viral expression in milk and transmission of infection.

Quantitative determinations of gp52, the main envelope glycoprotein, and p28, the main core protein, of MMTV, have been performed in about 1000 individual samples of milk of breeding females from our colony of MMTV-infected Swiss mice, a line characterized by a moderate incidence of mammary tumors. A computer analysis of the results showed: 1-- an important individual variation, ranging from 0 to 120 micrograms per ml of milk for p28, and from 0 to 320 micrograms per ml of milk for gp52; 2-- a variation of the release of both antigens during a single lactation, with a maximum on the 7--8th day of nursing; 3-- an increase of the release of both antigens with parity up to the 6th lactation, followed by a marked decrease during later lactations; 4-- a higher degree of infection in the offspring of 2nd and 3rd litters. The possible dependence of viral expression and transmission of infection upon factors such as cyclic activity of the mammary gland and progressive immunization of mice against MMTV is analyzed. The status of our laboratory line of MMTV infected Swiss mice is discussed in comparison with high and low tumor incidence strains.

In vitro cross-linking of bovine lens proteins photosensitized by promazines.

Promazine derivatives induce cross-linking of bovine lens crystallins in vitro by irradiation with near-ultraviolet (UV) light in the presence of O2, as revealed by electrophoresis after denaturation. With the five derivatives tested (promazine [PZ], chlorpromazine [CPZ], triflupromazine [ TFPZ ], methoxypromazine [ MTPZ ], and acepromazine [ ACPZ ] ), single-hit kinetics are observed. Evidence implicating the cation radicals of the PZ derivatives as the causative agent of this in vitro effect is presented. Hydroxyl radicals do not appear to be involved in the photo-cross-linking reaction. Sodium ascorbate protects against damage induced either by PZ derivatives plus light or by PZ cation radicals in the dark. These findings are discussed with respect to development of cataracts induced by these drugs in vivo.

Photodynamic activity of acridine orange: peroxide radical induction in DNA and synthetic polynucleotides.

As shown by electron paramagnetic resonance, acridine orange induces the formation of peroxide radicals in DNA when dye-DNA mixtures frozen at 77 K are irradiated with visible light. The reaction is oxygen dependent and strongly reduced by the addition of an electron scavenger. Factors of the medium can modulate the reaction: an ionic strength increased up to 0.3 greatly enhances the dye efficiency whereas the presence of phosphate ions has an inhibiting influence. Acridine orange, which is slightly less efficient than proflavine on native DNA, induces an important peroxide radical formation in poly(dG).poly(dC) but has no action on the poly(dA).poly(dT)polymer.

Terminations of DNA synthesis on 'proflavine and light'-treated phi X174 single-stranded DNA.

Bacteriophage phi X174 single-stranded DNA molecules were primed with five different restriction fragments and irradiated with visible light in the presence of proflavine. This photodamaged DNA was used as template for the in vitro complementary chain synthesis by E. coli DNA polymerase I (Klenow fragment). Chain terminations were observed by polyacrylamide gel electrophoresis of the synthesized products and localized by comparison with standard sequencing performed simultaneously on the untreated template. 90% of the chain terminations occurred one nucleotide before a guanine residue in the template strand. More than 80% of the sequenced guanine residues were blocking lesions demonstrating the absence of 'hot-spots' for the photodamaging effect of proflavine. At a defined position, the chain termination frequency increased linearly with the irradiation time and was directly influenced by the proflavine concentration present. An important part of lesions resulted from the action of singlet oxygen produced by excited proflavine as shown by the effect that both NaN3 and 2H2O exerted on the reaction. The induced blocking lesions must be important in vivo since no complete replicative forms could be extracted from cell infected with bacteriophages inactivated by 'proflavine and light' treatment.

Peroral infection of suckling mice with milk-borne mouse mammary tumour virus: uptake of the main viral antigens by the gut.

Persistence of mouse mammary tumour virus (MMTV) components in the digestive tract of suckling mice was investigated by immunoperoxidase staining of the main viral antigens and micro-immunoenzyme assays of gp52 and p28; these latter assays were also performed after ingestion of milk enriched in viral antigens using Cr2O3 as a marker for the alimentary bolus migration. When compared to the ingested antigens, the amounts of gp52 and p28 decreased during transit, p28 being more rapidly digested than gp52. The antigens were, however, destroyed to a much larger extent in the gut of the adult than in that of the newborn mouse. A fraction of the marker remained for a long time in the stomach; a prolonged retention was also observed with gp52 and especially with p28. Significant amounts of viral antigens were detected in the intestinal walls: both p28 and gp52 were found in the duodenum and small intestine. Moreover, the four viral antigens gp52, gp36, p28 and p8 were clearly observed in very large supranuclear vacuoles inside the epithelial cells of the distal part of the gut. Total particles can reach the intestine; the viral material could then be either destroyed or taken up in the epithelial cells by endocytosis, so that the intestinal epithelium might serve as a portal of entry for MMTV in the suckling mouse.