Folate hydrolase-1 (FOLH1) is a novel target for antibody-based brachytherapy in Merkel cell carcinoma.

BACKGROUNDS: Folate Hydrolase-1 (FOLH1; PSMA) is a type II transmembrane protein, luminally expressed by solid tumour neo-vasculature. Monoclonal antibody (mAb), J591, is a vehicle for mAb-based brachytherapy in FOLH1+ cancers. Brachytherapy is a form of radiotherapy that involves placing a radioactive material a short distance from the target tissue (e.g., on the skin or internally); brachytherapy is commonly accomplished with the use of catheters, needles, metal seeds and antibody or small peptide conjugates. Herein, FOLH1 expression in primary (p) and metastatic (m) Merkel cell carcinoma (MCC) is characterized to determine its targeting potential for J591-brachytherapy. MATERIALS & METHODS: Paraffin sections from pMCC and mMCC were evaluated by immunohistochemistry for FOLH1. Monte Carlo simulation was performed using the physical properties of conjugated radioisotope lutetium-177. Kaplan-Meier survival curves were calculated based on patient outcome data and FOLH1 expression. RESULTS: Eighty-one MCC tumours were evaluated. 67% (54/81) of all cases, 77% (24/31) pMCC and 60% (30/50) mMCC tumours were FOLH1+. Monte Carlo simulation showed highly localized ionizing tracks of electrons emitted from the targeted neo-vessel. 42% (34/81) of patients with FOLH1+/- MCC had available survival data f or analysis. No significant differences in our limited data set were detected based on FOLH1 status (p = 0.4718; p = 0.6470), staining intensity score (p = 0.6966; p = 0.9841) or by grouping staining intensity scores (- and + vs. ++, +++, +++) (p = 0.8022; p = 0.8496) for MCC-specific survival or recurrence free survival, respectively. CONCLUSIONS: We report the first evidence of prevalent FOLH1 expression within MCC-associated neo-vessels, in 60-77% of patients in a large MCC cohort. Given this data, and the need for alternatives to immune therapies it is appropriate to explore the safety and efficacy o f FOLH1-targeted brachytherapy for MCC.

Higher-order structure of mammalian chromatin deduced from viscoelastometry data.

The results of viscoelastometry (VE) for mammalian DNA have been puzzling because they have two orders of magnitude smaller measured viscoelastic relaxation times for mammalian chromosomes than that expected for DNA linear coils of chromosomal size. In an attempt to resolve this discrepancy, we have applied a recent model of G1 chromosome structure (J.Y. Ostashevsky, Mol Biol. Cell 9, 3031-3040, 1998) in which the 30 nm chromatin fiber of each chromosome forms a string of loop clusters (micelles). This model has two parameters: the number of loops per micelle (f) and the average loop size (Mf), which can be estimated independently from VE data. Using our VE data for plateau phase V79 Chinese hamster cells (unirradiated and X-irradiated with doses up to 40 Gy) we show that f approximately 13 , which is close to other estimates made using the model (f ranges from 10-20), and Mf approximately 2 Mbp, which is similar to estimates made from our nucleoid data (1.3 Mbp) and to estimates made in the literature using a variety of techniques (1-3 Mbp).

Increasing radiosensitivity in the course of fractionated X irradiation: the effect of contact with dead and dying cells.

We have measured survival after successive 2-Gy doses of X rays in HeLa cells and 1-Gy doses in cells of the nonimmortalized human fibroblast cell line AG15-22 under conditions where any effect of cell proliferation during multifraction X irradiation has been factored out. When HeLa cells in parallel series of (pseudo)hybrid spheroids (i.e. in agglomerates consisting of a mixture of supralethally irradiated HeLa feeder and viable HeLa cells) were exposed to n daily radiation doses and then trypsinized and exposed to the last dose, the surviving fraction at 2 Gy (SF2) declined exponentially from 0.55 +/- 0.01 to 0.31 +/- 0.01 after the fifth fraction. In monolayer HeLa cell cultures, the decline in SF2 was smaller but significant and was not influenced by the presence of feeder cells. Pure spheroids, composed entirely of viable HeLa cells, showed the same decline in SF2 as did monolayer cells. The cumulative-effect curve (i.e. the product of SF2 values) was linear-quadratic with the quadratic term increasing in the order monolayer, pure spheroids, pseudohybrid spheroids. SF2n and D0Eff (deduced from the initial SF2) severely underestimated cumulative radiosensitivity. This cumulative effect is clearly associated with the proximity of lethally irradiated cells and might be explained by differential population shifts in the course of the multifraction regimen. Similarly, AG15-22 cells irradiated with daily 1-Gy doses of X rays showed a larger increase in radiosensitivity when in hybrid spheroids than when in pure spheroids. However, for the AG15-22 cells, SF1 was twofold lower for the former than for the latter condition and remained constant for both conditions rather than decreasing with increasing fraction number. This large radiosensitizing effect remains unexplained.

Clonogenic inactivation of colon cancer-derived cells treated with 5-fluorouracil and indomethacin in hybrid spheroids.

The clonogenic hybrid spheroid assay has been used to determine the toxicity of 5-fluorouracil (5-FU), alone or in combination with indomethacin, in LoVo cells (a human colon adenocarcinoma line). The principal finding was that 5-FU toxicity, determined as loss of colony-forming ability, increased as a function of dose (concentration x duration of exposure), and that indomethacin causes a generalized alleviation of 5-FU toxicity, but only if given concurrently with 5-FU. The implications of these findings in the control of cancer cells by 5-FU are discussed.

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site.

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt[c]). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154-->Asp and Glu-154-->Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt(c) activity toward LexA have little or no effect on the coprt(c) activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.

Tests of the double-strand break, lethal-potentially lethal and repair-misrepair models for mammalian cell survival using data for survival as a function of delayed-plating interval for log-phase Chinese hamster V79 cells.

Our data (Reddy et al., Radiat. Res. 141, 252-258, 1995) on the kinetics of the repair of potentially lethal damage in log-phase Chinese hamster V79 cells are used to test some predictions which arise from the different assumptions of the repair-misrepair (RMR) (C. A. Tobias, Radiat. Res. 104, S77-S95, 1985), lethal-potentially lethal (LPL) (S. B. Curtis, Radiat. Res. 106, 252-270, 1986) and double-strand break (DSB) (J. Y. Ostashevsky, Radiat. Res. 118, 437-466, 1989) models. The LPL model defines the time available for repair of PLD (t(rep)) as the time taken to reach maximal survival in a delayed-plating recovery experiment. Those data show that after this time has elapsed, contrary to the expectation of the LPL model, survival can be increased by changing the medium used for delayed plating from fresh growth medium to conditioned medium. According to the RMR model, all potentially lethal lesions should also be committed by that time and be unavailable for repair in the new medium. Only the DSB model correctly predicted that PLD (= DSBs) would still be available for repair after that time. Second, data for split-dose recovery are used to predict the first-order kinetics time constant for DSB repair (tau(DSBR)) using the DSB model (24 +/- 1.5 min). This value is nearly identical to the value of 27 +/- 1 min determined from the data obtained by Cheong et al. using pulsed-field gel electrophoresis (PFGE) (Mutat. Res. 274, 111-122, 1992). The value based on PFGE is used to calculate the value of t(rep) predicted by the DSB model (2.6 +/- 0.1 h), which agrees with the value determined experimentally as the time when changing the delayed-plating medium from growth medium to conditioned medium no longer gives the full recovery seen with delayed plating in conditioned medium (2.5 h). However, some recovery was seen for a change in the medium (growth medium to conditioned medium) up to 5-6 h postirradiation. Reanalysis of the original data on DSB repair shows that they are consistent with two first-order repair rates (18 +/- 7 min and about 52 min). These results are consistent with two pools of DSBs (or cells), each with their own t(rep). The early t(rep), associated with tau(fast), is predicted to be 1.7 +/- 0.7 h, and the late t(rep), associated with tau(slow), is predicted to be about 5 h. Both values are in excellent agreement with the times at which changing from growth medium to conditioned medium no longer gives the full recovery seen in conditioned medium only (the early t(rep)), and the time when changing from growth medium to conditioned medium produces no further increase in survival (the late t(rep)), respectively. It is noted that attempts to correlate radiosensitivity with the rates of DSB repair, rather than using an explicit model such as the DSB model, are unlikely to be productive since survival depends on both tau(DSBR) and t(rep) (as defined in the DSB model) and the latter may be the more important determinant of radiosensitivity (as it appears to be for ataxia telangiectasia cells compared to normal fibroblasts and for irs compared to V79 cells).

Nutrient dilution before and after X irradiation increases the radioresistance of log- and plateau-phase Chinese hamster V79 cells.

Previous observations have shown that cells cultured in standard growth medium (100%) demonstrated similarly enhanced survival when incubated postirradiation either in non-growth-promoting conditioned medium or in growth-promoting 40% growth medium (Reddy and Lange, Radiat, Res. 119, 338-347, 1989). From these results, it was suggested that nutrient dilution altered radiosensitivity by a mechanism independent of progression of cells through the cell cycle. In this study, we have examined the effects on radiosensitivity of incubation in 40% growth medium prior to irradiation on both log- and plateau-phase Chinese hamster V79 cells and the effects on the distribution of cells in the cell cycle of incubation in 40% or 100% growth medium before and after irradiation. Radioresistance increased by a factor of 1.5-1.6 compared to 100% growth medium for both log-phase and plateau-phase cells cultured in 40% growth medium prior to X irradiation and incubated in either 40% growth medium or conditioned medium after X irradiation. The cell cycle distributions of log-phase cells in 100% and 40% growth medium before irradiation were identical. The change in cell cycle distribution induced by 10 Gy did not differ among log-phase cells incubated for 3 h postirradiation in 100% growth medium, 40% growth medium or conditioned medium. These results, in addition to supporting our previous conclusions, demonstrate that culturing prior to irradiation in 40% growth medium alone increases cell survival and that incubation in 40% growth medium before and after irradiation maximizes the survival of V79 cells.

Extracellular matrix (ECM) and cytoskeletal modulation of cellular radiosensitivity.

Modulation of radiosensitivity by components of the extracellular matrix (ECM) and cytoskeletal elements has not been adequately studied. Although differences in the radiosensitivities of cells grown as monolayers, as spheroids, or grown in vitro in animal models are known, explanations have in the past neglected possible influences by the ECM and cytoskeleton. Using collagen gel cultures, it is shown that the fibrillar component of the ECM (which is responsible for cell anchorage) induces shifts in radiosensitivity. The effect is critically dependent on the affinity of the cell type towards collagen. The shifts in radiosensitivity induced by ECM alteration are manifested as changed Dq values. By applying four specific cytoskeletal poisons which either stabilize or destabilize specific cytoskeletal elements, the involvement of microfilaments and microtubuli was qualitatively appraised. Cytochalasin B, which destabilizes microfilaments (by preventing polymerization), caused a significant rise in radioresistance. This rise was due to increased D0. Although the cellular morphological change accompanying cytochalasin B treatment was essentially similar to that obtained with trypsin, the respective shifts in radioresponses were qualitatively different and opposite, suggesting differences in mechanism of action.

Modulation of the effect of camptothecin in x-irradiated L5178Y-R and L5178Y-S cells by benzamide.

The L5178Y (LY) murine lymphoma subline, LY-R, is more radioresistant and more sensitive to camptothecin (CPT, inhibitor of topisomerase I) than the second subline used in our investigation, LY-S. Post-irradiation treatment with 3 microM CPT enhanced the radiosensitivity of LY-S cells (D0 decrease from 0.52 to 0.34 Gy), but did not change it in LY-R cells. Treatment with 2 mM benzamide [BZ, inhibitor of poly (ADP-ribosylation)] before x-rays and CPT increased the radiosensitivity of LY-R cells (D0 decrease from 1.15 to 0.52) without further modification of radiosensitivity of LY-S cells. Activity of topoisomerase I was diminished 10 min after x-irradiation (5 Gy) in LY-S, but not in LY-R cells. The data on DNA damage (fluorescent halo or comet assays) showed that the ultimate fate of the cells did not depend on the DNA damage pattern estimated immediately after treatment (e.g. the damage was greater in x-rays plus CPT than in BZ plus x-rays plus CPT treated LY-R cells, although the radiosensitivity was less). Aphidicolin (inhibitor of DNA polymerases alpha and delta) applied concomitantly with CPT in cells not pretreated with BZ prevented the increase in DNA damage in LY-R cells, but was without effect in LY-S cells. Taking into account the differential inhibition by x-rays of DNA synthesis in LY sublines and its reversion by BZ in LY-S but not in LY-R cells, we conclude that the pattern of DNA damage observed by the methods applied depended on the status of DNA replication.

Effect of methylation on the electrophoretic mobility of chromosomal DNA in pulsed-field agarose gels.

Factors other than molecular weight are known to affect DNA electrophoretic mobility. DNA methylation has been found to affect the curvature of DNA, causing anomalous mobility in polyacrylamide gels; the effect of methylation on the mobility of large DNA molecules in agarose gels was unknown. Chromosomal DNA from Mycoplasma capricolum, a wall-less prokaryote which has a low intrinsic methylation rate, was methylated in agarose blocks by SssI methylase, a de novo methylase with a CpG recognition sequence. (A surprising finding was that SssI methylase altered the structure of InCert, but not SeaKem Gold, agarose.) Restriction enzyme analysis was used to estimate the extent of CpG methylation. DNA methylation was found to have no effect on the electrophoretic mobility of full-length chromosomal DNA (1,120 kbp) in agarose gels. Therefore, methylation is not a source of error in PFGE-based size estimation for chromosomal DNA molecules less than 1.12 Mbp in agarose gels.

Content of iron and copper in the nuclei and induction of pH 9-labile lesions in L5178Y sublines inversely cross-sensitive to H2O2 and x-rays.

Cells from the L5178Y murine lymphoma subline LY-R are twice as resistant to killing by ionizing radiation than the subline LY-S. In contrast, LY-R cells are more sensitive to killing by H2O2, the effect being more pronounced at 37 degrees C than 0 degree C. Initial DNA damage after H2O2 treatment (both temperatures, 5 min) has been estimated by the 'comet' assay (single-cell gel electrophoresis) and fluorescent halo technique. According to both methods, the initial damage is significantly higher in LY-R cells, particularly that inflicted at 0 degree C. Differences between DNA unwinding and rewinding abilities at pH 9 and 6.9 (estimated by the fluorescent halo technique) point to a considerable difference in pH-9-labile damage between the sublines, as observed previously for x-irradiated cells (Kapiszewska et al. 1992). In contrast to findings with x-irradiated cells, however, after H2O2 treatment this damage is more extensive in LY-R cells than in LY-S cells. Thus, the initial pH-9-labile damage corresponds to the pattern of sensitivity to H2O2 and x-rays. We suggest that this is caused by different proportions of cuprous and ferric ions found in the nuclei of LY sublines and by the different ability of these ions to react with H2O2 and water radiolysis products. The copper/iron ratio in the nucleus is 1.31 in LY-R cells and 4.84 in LY-S cells.

Chinese hamster V79 cells harbor potentially lethal damage which is neither fixed nor repaired for long times after attaining maximal survival under growth conditions.

The kinetics of the repair and fixation of potentially lethal damage (PLD) was studied in log-phase Chinese hamster V79 cells. The postirradiation (10 Gy) survival of cells treated with hypertonic saline increased when these cells were incubated further in conditioned medium but not in growth medium, indicating that damage which is neither fixed by hypertonic saline nor amenable to repair in growth medium is nonetheless repaired in conditioned medium. Recovery of X-irradiated cells incubated in growth medium or in conditioned medium was maximal by about 70 min and was two times higher in conditioned medium than in growth medium. Cells incubated in growth medium for 70-120 min postirradiation continued to repair damage when subsequently shifted to conditioned medium and attained the same survival as that of cells in conditioned medium only. Thus PLD is not fixed by the time the recovery plateau has been attained in growth medium, and this unfixed PLD can still be repaired when cells are shifted to conditioned medium. To study the kinetics of fixation of PLD (without hypertonic saline), the survival of cells incubated in growth medium for up to 9 h postirradiation was compared with that for cells incubated in growth medium for different times followed by incubation in conditioned medium. These results show that the damage was neither fixed nor misrepaired in growth medium but rather remained unrepaired for up to 2 h, and that damage fixation in growth medium does not begin until after 2 h and is completed by 6 h postirradiation.

The 30 nm chromatin fiber as a flexible polymer.

Our analysis of the data of van den Engh, Sachs, and Trask (Science 257, 1410 (1992)), for the dependence of the mean square distance between pairs of hybridization sites (< L2n >, micron 2) on the known genomic distance (n, bp) separating these sites on chromosome number 4 in G1 human fibroblast nuclei, shows that < L2n > is proportional to n2v with v = 3/5 for n < 1 Mbp. The v-value of 3/5 is characteristic of flexible polymer chains with excluded volume effects in dilute good solutions. Since the DNA concentration in nuclei is very high (ca. 1-10 mg/ml), and theory (Flory, J. Chem. Phys. 17, 303, 1949) predicts v = 1/2 for overlapping polymers, the finding of v = 3/5 means that the chromatin fibers do not overlap in interphase nuclei. The dependence of < L2n > on n for n < 4 Mbp is consistent with the model of large (approximately 6 Mbp, 3 microns diameter) loops of interphase chromatin attached to nuclear membrane sites. Using the constant (e.g., Widom, Ann. Rev. Biophys. Biophys. Chem. 18, 365 (1989)) and variable (Williams & Langmore, Biophys. J. 59, 606 (1991)) diameter fiber models, the Kuhn statistical segment of the 30 nm chromatin fiber was estimated to have a length of 196-272 nm with a corresponding DNA content of 21-37 kbp.(ABSTRACT TRUNCATED AT 250 WORDS)

Is there a cell-to-cell contact effect on the X-ray dose-survival response of mammalian cells?

While a cell-to-cell contact effect has been reported for a Chinese hamster subline V79-171B, this was not observed for another subline V79 171-S. Therefore, we tested whether the cell-to-cell contact effect on cell survival depended on the cell line or the experimental conditions used. We have cultured and compared both sublines under identical conditions. Both sublines, cultured in Eagle's minimal essential medium (MEM) with 15% serum, had nearly identical cell doubling times and radiosensitivities. For both sublines, the survival of spheroid and monolayer cells subcultured immediately after irradiation were nearly the same, i.e., a radio-protective contact effect for spheroid cells was absent. Under conditions favorable for the repair of radiation induced damage, cell survival was higher for cells in monolayers than for cells in spheroids. Potentially lethal damage (PLD) repair and sublethal damage (SLD) repair were present in both sublines. However, the magnitude of expression of PLD by hypertonic saline was higher for monolayer than for spheroid cells. We conclude that: 1) the reported differences between V79 sublines (contact effect on survival) appear to be dependent on differences between experimental conditions rather than on cell type; 2) delayed plating technique does not detect PLD repair in round spheroid cells; and 3) detection of repair by split dose is independent of cell shape and/or two- or three-dimensional culture conditions.

Damage at two levels of DNA folding measured by fluorescent halo technique in X-irradiated L5178Y-R and L5178Y-S cells. II. Repair.

In the preceding paper we described the properties of nucleoids analyzed with the fluorescent halo assay at pH 6.9 and 9, as well as in the presence of reducing and chelating agents and after X-irradiation. We found analogies between the properties of type I and II nucleoids, as examined by Lebkowski and Laemmli (1982), and nucleoids analyzed with the fluorescent halo assay. We concluded that radiation-inflicted damage at two levels of DNA folding is measured at pH 6.9 and 9. In this paper we examined repair of damage to the nucleoid structure as assayed by the fluorescent halo method in X-irradiated L5178Y (LY) sublines; R (radiation resistant, D0 = 1.4 Gy) and S (radiation sensitive, D0 = 0.5 Gy). Halo diameters were measured after cell lysis in the presence of propidium iodide (PI; 0.5 to 50 micrograms/ml) at pH 6.9 and 9. The ability of DNA to be rewound at 10-50 micrograms/ml of PI was impaired by X-irradiation and partly restored during 90-min post-irradiation incubation, indicating damage to the superhelical structure and its partial restoration. The exponential time constants for repair were 10.1 min (LY-S, 6 Gy), 11.2 min (LY-R, 12 Gy), and 20.3 min (LY-s, 12 Gy) when measured at pH 9. In X-irradiated (12 Gy) LY-S cells, slower restoration of DNA supercoiling was observed at pH9 than at pH 6.9. The presence of labile lesions at pH 9 did not prevent restoration of the higher-order DNA structure, as estimated from DNA rewinding at pH 6.9 in LY-S cells.

Reduced sensitivity to camptothecin of topoisomerase I from a L5178Y mouse lymphoma subline sensitive to X-radiation.

Murine L517BY (LY) lymphoma sublines, LY-R (X-radiation resistant) and LY-S (X-radiation sensitive) displayed a difference in susceptibility to camptothecin: susceptibility of LY-S cells to the alkaloid was shifted towards higher concentrations as compared to LY-R cells. A similar difference was observed at the level of genomic DNA when a number of DNA-protein cross-links was determined or single-strand breaks were revealed by the fluorescent nucleoid halo assay. Activities of topoisomerases I and II were the same in both sublines. In turn, a higher resistance to camptothecin was found for the isolated LY-S topoisomerase I in the DNA cleavage test, suggesting that an altered enzyme was responsible for the susceptibility difference observed at the cellular level. In the relaxation test the enzymes from the two sublines showed a different sensitivity to beta-lapachone, an activator of topoisomerase I, but were similarly sensitive to all inhibitors, except camptothecin.

Response of primary colon cancer cells in hybrid spheroids to 5-fluorouracil.

We have measured the clonogenic survival of cells isolated directly from colon cancer surgical specimens and treated with 5-fluorouracil (5-FU). Enzymatically disassociated cells were incorporated into hybrid spheroids, consisting predominantly of nonproliferating HeLa feeder cells. Aliquots were exposed for 1.5 hr to a range of concentrations of 5-FU. From the decrease in clonogenic survival, as deduced from the frequency of colony formers among hybrid spheroids after chemical treatment, we were able to construct survival curves in 50% of the surgical specimens tested. A striking revelation was the presence of a resistant plateau in the survival curves, reminiscent of the solid tumor response to treatment with 5-FU. This resistance was absent in monolayer cultures. Evidence is presented that this resistance is due to the absence of, or delay in, cell cycle progression of cells residing in hybrid spheroids.

Potentiation of radiation lethality in mouse melanoma cells by mild hyperthermia and chloroquine.

To test the hypothesis that radiosensitization by combined mild hyperthermia and chloroquine may be increased by the presence of melanin in treated cells, Cloudman melanotic mouse melanoma S91/6 cells, and the amelanotic S91/amel cells were incubated during a 3 h post-irradiation period with 0.03 mM chloroquine at 41 degrees C. A considerable increase in radiation lethality was observed (radiation potentiation factor > 1.6) in both cases. Addition of 0.1 mM isobutyl-methyl xanthine (IBMX), a promoter of melanin synthesis, to the growth medium of S91/6 cells 10 days before irradiation, did not further increase the lethality of radiation followed by combined heat and chloroquine treatment. Under these conditions, toxicity to unirradiated cells was slight. On the other hand, 10 microM chloroquine showed similar toxicity to unirradiated B-16 mouse melanoma cells, but did not increase radiation lethality. Factors other than melanin content therefore play a role in the potentiation of radiation lethality by mild hyperthermia and chloroquine.