MSH4 acts in conjunction with MLH1 during mammalian meiosis.

MSH4 is a meiosis-specific MutS homolog. In yeast, it is required for reciprocal recombination and proper segregation of homologous chromosomes at meiosis I. MLH1 (MutL homolog 1) facilitates both mismatch repair and crossing over during meiosis in yeast. Germ-line mutations in the MLH1 human gene are responsible for hereditary nonpolyposis cancer, but the analysis of MLH1-deficient mice has revealed that MLH1 is also required for reciprocal recombination in mammals. Here we show that hMSH4 interacts with hMLH1. The two proteins are coimmunoprecipitated regardless of the presence of DNA or ATP, suggesting that the interaction does not require the binding of MSH4 to DNA. The domain of hMSH4 responsible for the interaction is in the amino-terminal part of the protein whereas the region that contains the ATP binding site and helix-turn-helix motif does not bind to hMLH1. Immunolocalization analysis shows that MSH4 is present at sites along the synaptonemal complex as soon as homologous chromosomes synapse. The number of MSH4 foci decreases gradually as pachynema progresses. During this transition, MLH1 foci begin to appear and colocalize with MSH4. These results suggest that MSH4 is first required for chromosome synapsis and that this MutS homologue is involved later with MLH1 in meiotic reciprocal recombination.

Comparative mapping of two adjacent regions of MMU19 with their human counterpart on HSA11q13.

High resolution physical maps of two adjacent regions of MMU19 were constructed in order to establish a comparative map between the pericentromeric region of MMU19 and its human counterpart on HSA11q13. These two physical maps span 2.5 and 0.5 megabases on MMU19. Long range restriction analysis and YAC contigs have been built, five genes were located on MMU19 and eight new STSs were generated. The 0.5-Mb map which has been positioned close to the centromere of MMU19, based on dual-color FISH experiments and genetic data, includes eight genes (Type I markers), three microsatellites (Type II markers) and five new STSs. The 2.5-Mb map is located more telomeric and contains seven genes, four microsatellites and four new STSs. Gene order and physical distances appear to be similar in human and in mouse in this 2.5-Mb region. Strikingly, the 0.5-Mb region has a similar size in human but gene order is shuffled. The overall comparative map shows that these two regions are inverted on MMU19 when compared with HSA11q13.

Expression and chromosomal localization of the Requiem gene.

Apoptosis in murine myeloid cell lines requires the expression of the Requiem gene, which encodes a putative zinc finger protein. We detected the protein in both cytoplasmic and nuclear subcellular fractions of murine myeloid cells and human K562 leukemia cells, which suggests that the protein might have a function distinct from a transcription factor. This distribution did not alter upon apoptosis induction by IL-3 deprivation. As an approach to investigate its role in development, we determined the spatio-temporal expression pattern in the mouse. Expression was detected in various tissues in earlier gestational age; however, confined to testes, spleen, thymus, and part of the hippocampus in the adult mouse. The expression profile is consistent with a functional role during rapid growth and cell turnover, and in agreement with a regulatory function for hematopoietic cells. The human cDNA clone sequenced showed high homology to its murine counterpart and extended the open reading frame by 20 codons upstream. The gene is located in the proximal region of mouse Chromosome (Chr) 19. In the homologous human region at 11q13, it is located at about 150 kb centromeric from MLK3.

Does the direct measurement of oxygen tension in tumors have any adverse effects?

A new polarographic histograph was tested in tumors to assess its potential adverse effects in mice. The tumor cell lines used were: the lewis lung carcinoma (3LL) and two human xenografts (Na11+ and HRT18). The C57BL/6 mice survival and number of pulmonary metastases were not altered after pO2 measurements. Whatever the cell line, tumor doubling times were not changed after pO2 measurements. The new polarographic histograph was found to be reliable.

Radiosensitization by the combination of etanidazole (SR-2508) and pimonidazole (Ro 03-8799) in human tumor xenografts.

The two current second generation radiosensitizers SR-2508 (etanidazole) and Ro 03-8799 (pimonidazole) have different dose-limiting toxicities in humans. SR-2508 produces peripheral neuropathy, whereas Ro 03-8799 causes acute CNS toxicity. Overall toxicity can be minimized while increasing maximal radiosensitization by combining the two sensitizers. The additivity of their radiosensitizing properties was tested using clinically relevant SR-2508 and Ro 03-8799 concentrations in two human tumor xenografts: a rectal adenocarcinoma (HRT18) and a pigmented melanoma (Na11+). Drug concentrations were determined by a High Performance Liquid Chromatography method. Tumor response was assayed by clonogenic cell survival. The maximal radiosensitizing effect was determined using a single dose of radiation and high doses of drugs. For HRT18, the maximal radiosensitization was achieved when SR-2508 and Ro 03-8799 were given 45 and 30 min, respectively, before irradiation. For Na11+, the maximal radiosensitizing effect was lower than for HRT18 and not significant, but a trend was observed at about 30 min before irradiation for both drugs. Using clinically relevant doses, mean Sensitizing Enhancement Ratio (SER) values for HRT18 were: 1.3 (Ro 03-8799: 0.1 mg/gram body weight (gbw) or SR-2508: 0.2 mg/gbw), and 1.5 (Ro 03-8799: 0.1 mg/gbw + SR-2508: 0.2 mg/gbw). Mean SER values for Ro 03-8799 0.2 mg/gbw, SR-2508 0.4 mg/gbw, and Ro 03-8799 0.2 mg/gbw + SR-2508 0.4 mg/gbw were 1.5, 1.5, and 1.8, respectively. No significant radiosensitizing effect was observed on Na11+ with either drug administered singly or in combination and at the same concentrations. Our results suggest that the effectiveness of the two sensitizers administered alone or in combination may be tumor-dependent and that melanomas may not be good candidates.

The effect of hydralazine on blood flow and misonidazole toxicity in human tumour xenografts.

The effect of post-irradiation hypoxia induced by 5 or 30 mg/kg hydralazine has been studied in three human tumour xenografts (two rectocolic adenocarcinomas and one melanoma) treated with two doses of misonidazole similar to those used in patients (0.1 and 0.2 mg/g). Only a small sensitization was detected using an in vitro colony assay. These results are in marked contrast to the results obtained with rodent tumours. This difference between human tumour xenografts and rodent tumours might be explained by differences in the reduction of tumour blood flow after hydralazine administration (5 and/or 10 mg/kg). Using the laser Doppler technique, the tumour blood flow reduction was 33% and 25% of the control for NA11 and HRT18 tumours, respectively. In contrast, hydralazine induced a 60-70% reduction in blood flow in the murine SCCVII tumour. Using the fluorescent marker Hoechst 33342, the reduction in perfusion was again more pronounced in the murine tumour as compared to the Na11 and HRT18 xenografts. The differences between human tumour xenografts and rodent tumours are not linked to the mouse strain used (nude versus C3H) nor to a tumour bed effect.

Glutathione and cysteine levels in human tumour biopsies.

There is evidence that some human tumours could be treated with a combination of buthionine sulfoximine and hypoxic cell sensitizers. However, clinical application of this technique requires a prior knowledge of the level of non-protein bound sulfhydryl (NPSH) compounds in these tumours. The present study provides data on the levels of glutathione (GSH) and cysteine (CYS) in human tumour biopsies from the cervix and from the head and neck. The NPSH compounds were measured by high performance liquid chromatography. The median GSH values were 20.5 nmol/mg protein (cervix) and 23 nmol/mg protein (head and neck) while the median CYS values were 4.4 (cervix) and 4.2 nmol/mg protein (head and neck). The values varied widely from one patient to another.

Ro 03-8799: preferential relative uptake in human tumor xenografts compared to a murine tumor: comparison with SR-2508.

The pharmacokinetics of Ro 03-8799 and SR-2508 were studied in 6 tumor cell lines (5 human, 1 murine) transplanted into athymic nude mice. The human tumors were rectocolic adenocarcinomas (HRT18, HT29) and melanomas (Be11, Na11+, Na11-), the rodent tumor was a mammary tumor (EMT6). The concentrations of drugs in tumor, blood, plasma, and red cells were measured by HPLC 15, 30, and 45 minutes after the simultaneous i.v. injection of 0.1 mg/g of each compound. Little or no difference was found between the concentrations in the plasma, blood, and red cells for either drug; SR-2508 concentration was higher than that of Ro 03-8799. Both compounds were concentrated in all the tumors, the concentration increases or decreases as a function of the time, depending on the cell line; there was more Ro 03-8799 in the pigmented melanomas than in the other tumors. The relative uptake of Ro 03-8799 and SR-2508 in the tumor was evaluated by the ratio of drug concentrations in the tumor and in the blood. The results suggest that Ro 03-8799 and SR-2508 accumulated in all the tumors, the relative uptake of Ro 03-8799 was higher than that of SR-2508. For human tumors, the ratios increased between 15 and 45 minutes. They ranged from 1.2 to 3.6 for SR-2508 and from 3.1 to 14.6 for Ro 03-8799. For Ro 03-8799, the highest ratios were found for the melanomas; the uptake was higher in the pigmented Na11+ tumors than in the non-pigmented Na11-. The ratios for EMT6 were about 2.3 for SR-2508 and 4.7 for Ro 03-8799; these ratios varied slightly between 15 and 45 minutes.

SR-2508 plus buthionine sulfoximine or SR-2508 alone: effects on the radiation response and the glutathione content of a human tumor xenograft.

This study determined the radiosensitivity of the human tumor xenograft HT29 and its glutathione (GSH) and cysteine (CYS) contents after treatment with both buthionine sulfoximine (BSO) and SR-2508 or SR-2508 alone. Tumor radiosensitivity was assessed by the in vitro colony assay and thiol content was measured by high-performance liquid chromatography. The radiosensitizing effect of SR-2508 is dose dependent and increases when higher doses of radiation are given. SR-2508 given alone does not modify GSH and CYS content; however, when given with BSO, the GSH level is significantly reduced, yet radiosensitivity of the HT29 tumor is only slightly increased. These results have been compared to our previously observed results of HT29 treatment with misonidazole (MISO), BSO, or MISO + BSO.

Partial hypoxia as a cause of radioresistance in a human tumor xenograft: its influence illustrated by the sensitizing effect of misonidazole and hyperbaric oxygen.

While previous studies with three human tumor xenografts suggest that contact-resistance plays a major role in the response of these tumors to radiation, it remains possible that partial hypoxia may provide an alternate explanation. The present study was carried out to check this possibility by investigating the influence of misonidazole (MISO) and hyperbaric oxygen (HBO) on both the initial and distal components of the survival curves of HRT18 tumor cells. The effect of a challenge dose of radiation on the initial radioresistance of this tumor was also studied. To assess the effects of MISO and HBO, tumor cell survival was determined by excision assay in two groups of tumor-bearing mice, one given MISO (1 mg/g body weight, i.p.) 45 min before irradiation and the other exposed to HBO (3.5 bars). MISO treatment caused greater sensitization than HBO. The enhancement ratios at the 5.10(-1) level were 1.7 (MISO) and 1.7 (HBO); at the 10(-1) level, they were 1.6 (MISO) and 1.4 (HBO); while at 10(-2), they were 1.6 (MISO) and 1.4 (HBO). These two sensitizing effects favor the hypothesis that solid tumors contain a compartment of partially hypoxic cells. To study the effect of a challenge radiation dose on initial radioresistance, tumors were given a challenge dose of 8 Gy, followed 24-48 hr later by doses ranging from 2-12 Gy. The challenge dose did not modify the shape of the survival curve.

Radiobiological evaluation of a newly synthesized cysteamine derivative.

A new cysteamine-based compound, I109 (N-glycylglycyl-S acetylcysteamine trifluoroacetate) was tested on both normal tissues and tumors to evaluate its radioprotective potential. I109, which is three times less toxic than WR-2721, was injected at a dose approximately equal to half the LD50(30 days). The Protection Factor (PF = gamma ray dose ratio) after whole body irradiation was 1.3-1.4 for intestinal death and 1.4-1.5 for hemopoietic death when intervals of 40 or 20 min elapsed between injection and the end of irradiation. A crypt cell assay was done for both I109 and WR-2721; PFs were 1.1 and 1.4, respectively. I109 was then tested on five solid tumors. For each cell line (4 human, 1 murine) one dose of radiation was delivered. Surviving fraction ratios with and without drug ranged between 2.4 and 14.1 when an interval of 20 min elapsed between injection and the end of irradiation. The degree of radioprotection proved time dependent for EMT6 and HRT18; radioprotection afforded by WR-2721 on these tumors is either similar or greater than radioprotection afforded by I109 depending on the time interval between injection and irradiation.

Influence of buthionine sulfoximine and misonidazole on glutathione level and radiosensitivity of human tumor xenografts.

We have studied the effect of buthionine sulfoximine (BSO; a gamma-glutamylcysteine synthetase inhibitor) administration, either alone or combined with misonidazole (MISO), on five human tumor xenografts (three melanomas: Bell, Mall, and Nall; and two rectocolic adenocarcinomas: HT29 and HRT18) transplanted into mice. Two criteria were used, the nonprotein bound sulfhydryl (NPSH) level (glutathione (GSH) and cysteine (CYS] and the fraction of surviving tumor cells after gamma irradiation. GSH and CYS were estimated by HPLC and cell survival by in vivo-in vitro clonogenic assay. Administration of BSO alone (three injections of 10 mumol/g) prior to irradiation always produced a significant reduction in the GSH level while MISO administration (1 mg/g) did not consistently influence the NPSH level. While BSO had little or no radiosensitizing effect, MISO always induced radiosensitization (enhancement ratio between 1.6 and 1.8). This effect did not depend on the fraction of surviving hypoxic cells. An increase in MISO-induced radiosensitization produced by BSO was cell-line dependent. Results do not seem to support the hypothesis of a relationship between the GSH level at the time of irradiation and the radiosensitization induced by BSO or BSO + MISO. However, BSO treatment may not have been able to reduce endogenous thiols to a low enough level to test the hypothesis.

Radioprotection of EMT6 tumor by a new class of radioprotectors based on a pseudo-peptide cysteamine combination.

Although WR-2721 preferentially protects normal tissues against irradiation, it seemed desirable to find other drugs presenting a lower toxicity and the same radioprotective properties. A new compound, I 102, was selected; it was characterized on one hand by a coupling between cysteamine and an amino-acid, and on the other hand by an acetyl-group, which protects the thiol function. The effects of WR-2721 and of I 102 were studied on EMT6 tumors grafted on BALB/c mice. Whatever the size of the tumor, the cell survival increased as a function of the time elapsed between the injection of I 102 and the end of the irradiation (TI). In contrast, the radioprotection afforded by WR-2721 was found to be independent of TI. The survival curves suggest that, like WR-2721, I 102 protects essentially oxygenated cells.