Retinopathy induced in mice by targeted disruption of the rhodopsin gene.

Retinitis pigmentosa (RP) represents the most common mendelian degenerative retinopathy of man, involving death of rod photoreceptors, cone cell degeneration, retinal vessel attenuation and pigmentary deposits. The patient experiences night blindness, usually followed by progressive loss of visual field. Genetic linkage between an autosomal dominant RP locus and rhodopsin, the photoreactive pigment of the rod cells, led to the identification of mutations within the rhodopsin gene in both dominant and recessive forms of RP. To better understand the functional and structural role of rhodopsin in the normal retina and in the pathogenesis of retinal disease, we generated mice carrying a targeted disruption of the rhodopsin gene. Rho-/- mice do not elaborate rod outer segments, losing their photoreceptors over 3 months. There is no rod ERG response in 8-week-old animals. Rho+/- animals retain the majority of their photoreceptors although the inner and outer segments of these cells display some structural disorganization, the outer segments becoming shorter in older mice. These animals should provide a useful genetic background on which to express other mutant opsin transgenes, as well as a model to assess the therapeutic potential of re-introducing functional rhodopsin genes into degenerating retinal tissues.

Cell kinetics in human malignancies studied with in vivo administration of bromodeoxyuridine and flow cytometry.

Bromodeoxyuridine (BrdUrd) is a pyrimidine analogue which is incorporated into the DNA of proliferating cells. When in vivo BrdUrd infusion is coupled with bivariate flow cytometry to measure cell BrdUrd incorporation and DNA content, both the percentage of DNA-synthesizing cells [BrdUrd-labeling index (LI)] and the DNA synthesis time (TS) can be determined on the same tissue sample. From experimentally determined LI and TS, the potential doubling time of the population and its cell production rate are calculated. To ascertain whether the BrdUrd infusion method is clinically feasible and if data are reliable, we studied patients with leukemia, refractory anemia, multiple myeloma, and brain and gastric tumors. The BrdUrd incorporation data were compared with those determined on duplicate samples with the techniques conventionally used for LI and TS values, i.e., 3H- and 14C-labeled thymidine autoradiography, respectively. The complete BrdUrd procedure takes 6-9 h, and no immediate toxicity from BrdUrd administration has been observed. In an 8-month period, 154 patients were studied. Successful LI and TS determinations were obtained in 78.9 and 59.7% of cases, respectively, more often in hematological than in solid tumors. The values for LI and TS assessed with the BrdUrd technique were very close to those found with 3H- and 14C-labeled thymidine autoradiography (r = 0.88, P less than 0.005, and r = 0.89; P less than 0.005, respectively). The potential doubling time and production rate were accordingly similar. These data indicate that in vivo BrdUrd infusion coupled with flow cytometry measurements can be performed in clinical settings and that this method is reliable. It could be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and for planning radio- and/or chemotherapy.

Combined analysis of DNA ploidy index and N-myc genomic content in neuroblastoma.

The aim of the study was to assess, in a group of nonselected patients with neuroblastoma, the prognostic value of both N-myc gene amplification and DNA ploidy index, taking into account potential confounding factors such as age and stage. Of 59 patients studied, 23 were younger than 1 year at diagnosis, 31 presented with stage IV, 10 with stage III, 5 with stage II, 8 with stage I, and 4 with stage IV-S. N-myc genomic content was analyzed by Southern blot hybridization technique and N-myc amplification (greater than or equal to 3 copies/haploid genome) was present in 6 stage IV, 2 stage III, and 1 stage IV-S. The DNA ploidy index was analyzed by flow cytometry. Of the 59 neuroblastomas, 26 were diploid (DNA index, 1) and 33 were aneuploid (DNA index, greater than 1). The majority of the aneuploid tumors (28 of 33) were near-triploid with DNA indexes between 1.25 and 1.68, 4 were near-diploid (DNA index up to 1.18), and 1 was hypotetraploid (DNA index, 1.85). The proportion of near-triploid tumors was significantly greater among patients under 1 year of age and among patients presenting with stages I, II, and IV-S. Interestingly, 0 of 28 near-triploid neuroblastomas exhibited N-myc gene amplification, compared to 9 of 31 in the group of diploid, near-diploid, and hypotetraploid tumors (Fisher's exact test, P less than 0.001). Four factors were significantly related to a high risk of relapse in univariate analysis, i.e., age, stage, DNA index, and N-myc amplification. In multivariate analysis, only N-myc amplification and the DNA index remained significantly associated with a high risk of relapse. The 2-year disease-free survival rate was 94% (95% confidence interval, 77-98%) for patients with near-triploid neuroblastoma, compared to 45 and 11% (95% confidence interval, 32-70 and 4-23%) for patients with diploid or near-diploid tumors, without and with N-myc amplification, respectively. We concluded that the combination of N-myc and DNA index should be included in routine management of neuroblastoma.

Proliferative changes in two murine tumours in response to single or fractionated doses of X-rays.

Studies were carried out to investigate proliferative changes in two murine experimental tumours in response to radiation. Results were generated using bromodeoxyuridine labelling and flow cytometry. This study demonstrates the possible ambiguity of previous studies using tritiated thymidine in which inability to discriminate normal and tumour cell components in murine tumours may lead to different values for cell kinetic parameters. In particular, the sarcoma F appeared to have a growth fraction of 0.62 when all cells were considered; in reality the growth fraction of the tumour cells only (based on DNA content discrimination) was close to unity. Radiation, administered either as single or fractionated doses, caused little change in the proliferative characteristics of the sarcoma F tumour but had profound effects on the adenocarcinoma Rhodesia tumour. Major changes were the accumulation of cells in G2 for several days after the end of the radiation treatment in both tumours and a dramatic drop in labelling index of the Rhodesia tumour. In neither tumour was there any evidence to suggest an increase in tumour cell proliferation during or after the irradiations. The diploid cells within the sarcoma F tumour showed an initial depression of labelling index followed by a rapid increase overshooting the control labelling index at higher radiation doses. Much of the effects could be attributed to cell cycle delays.

Enhancement of chemotherapy agents.

The interaction in vivo between misonidazole and cytotoxic drugs is reviewed. In general MISO interacts best with bifunctional alkylating agents and nitrosoureas when there is generally a therapeutic gain at large MISO doses. The interaction is unlikely to be a result of pharmacokinetic effects of MISO, but may reflect its ability to inhibit recovery from drug induced damage. It is important to extend these studies to clinically relevant MISO doses simulating human pharmacokinetics.

A comparison of the ability of some radiosensitizers undergoing clinical trials to act as chemosensitizers.

The abilities of misonidazole, Ro 05-9963, Ro 03-8799 and Sr-2508 to enhance the action of the drugs cyclophosphamide (CY) and melphalan (L-PAM) have been compared in two mouse fibrosarcomas at acute and chronic sensitizer doses. SR-2508 was not effective with either CY or L-PAM in either tumor. Some enhancement of CY was obtained with Ro 05-9963 and Ro 03-8799; however, the degree of enhancement varied according to tumor and acute or chronic sensitizer dose. In all cases, the degree of enhancement was less than that obtained with an equivalent dose of misonidazole in both tumor systems. Of the four compounds tested, MISO would appear to have the most potential as a chemosensitizer.

Radiosensitization by misonidazole during recovery of cellular thiols following depletion by BSO or DEM.

V79 cells have been depleted of their endogenous thiols by treatment with 100 microM BSO for 16-18 hr, or 0.5 mM DEM for 1 hr. The recovery of cellular thiols after removal of the drugs was determined by h.p.l.c. or flow cytometry and the sensitizer enhancement ratio for 100 microM misonidazole was measured as a function of time after removal of the drugs. The SER of 1.2 for control (hypoxic) cells increased to 1.8 for BSO treated (hypoxic) cells and 2.2 for DEM treated ones, when thiol levels were below 10% of controls. The SER and thiol levels returned to control values within 5 hr of removing DEM. After BSO there was little change during the first 5 hr and then a gradual return to normal values by 24 hrs. The major fall in the SER after removal of the drugs occurred as the cellular thiols increased to 60% of control values.

The effect of prolonged high dose misonidazole on tumor response to radiation.

WHFIB and SA F tumors were exposed to misonidazole (MISO) concentrations of 2.5 mM or more for up to 4 hours (SA F) or 6 hours (WHFIB). There was no increase in the MISO enhancement ratio (SER) in the SA F for a 4 hour exposure to MISO relative to that following a single injection. In the WHFIB tumor, the ER increased from 2.2 for a single MISO injection to 2.5 for a 4 hour contact with MISO for tumor growth delay, and from 2.1 to 2.3 for a cloning assay. (These differences may not be statistically significant) Prolonged contact with MISO was toxic and reduced the body temperature by 4 to 5 degrees C. For WHFIB cells in vitro, when the contact time (in hypoxia) with 2.5 mM MISO was increased from 0.5 to 2.5 hours, the ER increased from 2.1 to 2.9 at 37 degrees C and from 1.9 to 2.5 at 33 degrees C.

Tumor cell kinetics, local tumor control, and accelerated radiotherapy: a preliminary report.

The local tumor control achieved in patients treated in a pilot study of continuous, hyperfractionated, accelerated radiotherapy has been related to the tumor cell kinetics evaluated by in vivo administration of bromodeoxyuridine and flow cytometry. In 42 of 50 patients with advanced squamous cell carcinomas in the head and neck region it was possible to sample the primary tumor prior to treatment. In three further cases, involved regional nodes were studied: in the remaining five, tissue was obtained subsequently either from a local recurrence or from a distant metastasis. Successful cell kinetic measurements were made in 38 (90%) of the 42 primary tumors. The median values of the labelling index, the duration of the DNA synthetic phase and, thus, the potential doubling time for all primaries were 7.1%, 9.8 hr, and 3.9 days, respectively. Complete regression was achieved in 28 (74%) of the primary tumors and in 23 (61%) this was maintained to the time of observation for this report. There was no significant influence of any of the cell kinetic parameters upon the immediate or longer term local tumor control. This result is compatible with the overcoming of cellular repopulation by the acceleration of radiotherapy.

Pharmacokinetic studies using multiple administration of RO 03-8799, a 2-nitroimidazole radiosensitizer.

It has been shown that the propanolamine 2-nitroimidazole Ro 03-8799, is about an order of magnitude more efficient than misonidazole as a radiosensitizer in vitro, which is more than its electron affinity would predict. However, in vivo studies have so far shown only a minimal improvement compared to misonidazole. One reason for this could be that diffusion to the site of action in the hypoxic cell is restricted by the short half-life in the mouse and the net positive charge carried by the drug at physiological pH. Ways of maintaining a constant blood or tumor concentration over a period of 1--2 hours were investigated so that the tumor response could be assessed after this time. Initial experiments used multiple intraperitoneal or intravenous injections, for which the amount of drug required can be calculated theoretically provided sufficient pharmacokinetic data are available. Later experiments using continuous infusion of the drug were aimed at maintaining the gross tumor concentration equal to that found at the time of peak sensitization following a single administration. Results will be presented using each of these approaches. The technical feasibility and advantages of each method, together with general problems associated with interpreting any radiobiological results obtained using this approach will be discussed.

The effect of cytotoxic drugs with or without misonidazole on leucopenia in three strains of mice.

Leucopenia is a major dose-limiting effect of many cytotoxic drugs. We have measured the effect of melphalan and four other drugs either alone, or with misonidazole on total white cell count in mice. Three strains were used, though not with all drugs. White cell counts were determined 3, 5, and 7 days after giving the cytotoxic drugs either with or without misonidazole. For all the drugs used, the nadir in white cell count was at 3 to 5 days when the dose-effect curves were approximately expotential. Only in the case of chlorambucil and mitomycin-C did misonidazole enhance the leucopenia. There was no enhancement of the effects of melphalan, cyclophosphamide or CCNU. In the case of mitomycin C the effect of misonidazole was to delay the recovery in the white cell count. It would appear that enhanced leucopenia from the combination of misonidazole with cytotoxic drugs may depend on the drug used. With three of the five drugs, the absence of an effect of misonidazole implies that any enhanced damage to tumors would represent a true increase in therapeutic effectiveness.

Cell proliferation and differentiation in squamous cancer.

Cellular differentiation is generally regarded as an important histological predictor of tumour behaviour. Verrucous carcinoma is a very well-differentiated tumour characterised by slow enlargement and an unsatisfactory response to radiotherapy. Tumour cell kinetic studies using the intravenous administration of bromodeoxyuridine (BrdUrd) before tumour sampling have shown these tumours to have high labelling indices and short duration of DNA synthesis. These values result in short potential cell doubling times of these tumours although they then have a well-differentiated appearance histologically. Study of 20 other squamous cell carcinomas has shown no relationship between grade and cell proliferation.

Radiation-induced cell cycle delay measured in two mouse tumors in vivo using bromodeoxyuridine.

The magnitude of the delay of cells in the phases of the cell cycle after irradiation may be related to the radioresponsiveness of tumor cell populations. In this study we have quantified division delay in two mouse tumors in vivo after single and fractionated doses of X rays and single doses of neutrons. The incorporation of bromodeoxyuridine and flow cytometry provided a sensitive and quantitative method to detect cell cycle perturbations after radiation treatment. The more rapidly growing SAF tumor showed less G2-phase delay per gray than a more slowly proliferating tumor, the Rh (0.9 vs 1.8 h). In addition, the SAF tumor failed to show any G1/S-phase delay while the Rh tumor experienced a longer G1-phase delay than that measured for G2 phase (3.1 vs 1.8 h). There was a trend in both tumors for lower doses to be more effective in producing cell cycle delays. Neutrons caused longer G2-phase delays on a unit dose basis, 2.5 and 5.4 h for the SAF and Rh tumors, respectively. The RBE for neutrons for division delay was found to be 2.9 and 2.8 for the SAF and Rh tumors, while the RBE for growth delay was 3.4 and 3.5. Fractionation of the X-ray dose caused a reduction in division delay at higher total doses (10 or 12 Gy) but was without effect at the lower dose studied (6 Gy). These studies show the feasibility of measuring cell cycle delays in vivo, and future developments are suggested for a possible predictive test in patients receiving radiotherapy.

Can cell kinetic parameters predict the response of tumours to radiotherapy?

Three potential predictive assays of the repopulation component in tumour response to therapy are considered. (1) The DNA index can easily be measured. It is of prognostic value for cancers of certain sites, aneuploidy being a bad prognostic indicator. It is not strictly an indicator of cell proliferation. (2) The in vitro labelling index is of predictive value in early stage operable breast cancer and in head and neck cancer. In the former a high pretreatment labelling index can identify patients who could benefit from adjuvant chemotherapy. (3) The tumour potential doubling time (Tpot) can be measured rapidly following in vivo labelling with bromodeoxyuridine or iododeoxyuridine. We have measured Tpot in over 100 solid tumours with a success rate of about 75 per cent. Nearly 50 per cent of the tumours have a pre-treatment potential doubling time of 5 days or less. These would be suitable candidates for accelerated fractionation.

Cell survival and DNA double-strand break repair following X-ray or neutron irradiation of V79 cells.

We have investigated the effects of 250kVp X-rays and 2.3 MeV (mean energy) neutrons on the cell survival, DNA double-strand break (dsb) induction and repair (using the Kohn neutral elution technique) in V79 cells. The lethal effects of neutrons were shown to be significantly greater than for a similar dose of X-rays (RBE = 3.55 at 10 per cent survival). However, the RBE for dsb induction, in a dose range of 10-50 Gy, was 1. On investigating the repair of DNA dsb induced by either X-ray or neutron irradiation, clear differences in the pattern of repair were found. Both a fast and a slow component of repair were seen in both cases; the former, however, was reduced following neutron irradiation and, since the amount of slow repair was similar in both cases, this resulted in proportionally more unrejoined breaks after neutrons. These experiments were carried out with elution buffer at pH 9.6; however, when similar experiments were performed at pH 7.2 the results obtained support our earlier findings. We suggest that these differences in DNA dsb repair reflect basic differences in the nature of the lesions induced by high- and low-LET ionizing radiations.

The rejoining of DNA double-strand breaks following irradiation with 238Pu alpha-particles: evidence for a fast component of repair as measured by neutral filter elution.

The induction and repair of DNA double-strand breaks (DSB) following exposure to 238Pu alpha-particles was examined in V79-379A cells. The technique of neutral filter elution was used in these investigations at both pH 9.6 and pH 7.2. The initial dsb yield was found to be similar to that seen after 250 kVp X-ray or 2.3 MeV neutron exposure. However, the pattern of dsb rejoining after alpha-particle irradiation did not follow that seen after X-rays or neutrons. A very fast initial component, complete within 2 min of incubation following irradiation, removed 70 per cent of the dsb seen after 40 Gy alpha-particles; very little slow rejoining was seen. This contrasts sharply with the dsb rejoining seen after X-ray or neutron exposure, and presumably reflects the differences in the nature of the dsb induced and the way they are repaired.

The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage.

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.

The problem of atopic eczema: aetiological clues from the environment and lifestyles.

Atopic eczema is the most common inflammatory skin disease in children, affecting around 10% of children in the developed world. It can be a distressing condition, influencing children's well-being, personal and educational development, and family life, and it has huge economic implications for health services and individual budgets. Like other atopic diseases such as asthma and hay fever, the prevalence of atopic eczema has increased substantially over the last 30 years, for reasons largely unknown. Although a genetic predisposition to the disease has been implicated, evidence from a range of sources suggests that environmental factors play a crucial role in the disease expression. This paper reviews the epidemiology of atopic eczema, with particular attention to potential environmental aetiological factors and draws evidence from studies in the UK and internationally. First, atopic eczema has been found to vary socially and to be more prevalent in the UK among social class I and II families than among other socio-economic groups. Second, it has been suggested that cross infection from other siblings in large families may have a protective role in atopic disease expression. Third, it has been proposed that an increased risk of atopic eczema may result from decreases in helminthic infestation. Fourth, studies of migrant groups have shown large increases in disease prevalence compared with migrants' country of origin, suggesting clues as to the importance of socio-economic and environmental changes such as those associated with industrialization. Finally, a distinct and consistent geographical pattern of eczema has been observed in the UK which cannot be explained by social class distribution. The various types of study have attempted to identify reasons for differences in prevalence but, to date, no definitive causation has been identified. In some cases, specific risk factors have been suggested and include house dust mites, dietary allergens and irritants. It is argued here that the aetiology is unlikely to be simple or uni-causal and that an understanding of the relationships between the disease and behaviour, lifestyle, home and external environmental factors is crucial. This paper reports the preliminary stages of an interdisciplinary research project involving dermatologists, epidemiologists and health geographers, and calls for investigation into associations between atopic eczema and possible environmental and lifestyle factors. These include behavioural factors, microenvironment factors and macroenvironments.

A comparison of the effects of radiation on tumour growth delay and cell survival. The effect of radiation quality.

Measurements have been made of the effects of 250 kV X-rays and cyclotron produced neutrons on the delay in growth of a rat fibrosarcoma (RIB5C) and on tumour cell survival assayed in vitro after irradiation in vivo. For doses above 300 rads of neutrons the RBE for cell survival was greaer than that for tumour growth delay. This may be due to X rays causing a greater delay in cell proliferation than neutrons for a given level of cell survival. This would be the opposite effect to that found by BBarendsen and Broerse (1969), irradiating a rat rhabdomyosarcoma. Another possibility is that meassurements of cellular radio-sensitivity which involve removal of cells from their normal environment may lead to incorrect estimates of cell survival in situ. A plot of the RBE for growth delay against the reciprocal of the neutron dose indicated that the dose level at which the RBE became dependent on the fraction of hypoxic tumour cells was larger than that for cell survival, indicating a smaller "effective" hypoxic fraction than that estimated from the cell-survival curves.

The effect of radiation on tumour growth delay, cell survival and cure of the animal using a single tumour system.

The response of a murine tumour to single doses of X rays has been measured using three different assays--animal cure, cell survival in vitro after irradiation in vivo, and tumour growth delay. The dose to cure 50% of the animals, the TCD50, was 79.0 Gy. This was not affected by clamping the tumours to render all the cells hypoxic at the time of irradiation, implying that most of the cells in the tumour were hypoxic already. The enhancement ratio for the hypoxic cell sensitizer Ro-07-0582 was 2.1. The cell survival assay gave an enhancement ratio of 1.6 and an hypoxic fraction of 5%. The discrepancy in the estimates of the hypoxic fraction can be explained by the ability of the naturally hypoxic cells, but not the oxic ones, to recover from potentially lethal damage in vivo. Neither the cell survival assay nor the growth delay assay agreed with the TCD50 assay as to the effect of the hypoxic cell sensitizer, even allowing for recovery from potentially lethal damage. It is doubtful whether the measured survival curve would predict the measured TCD50.