Differential retention of tumor- and differentiation-suppressor functions in cells derived from a human squamous cell carcinoma.

Three morphologically distinct cell lines--F.2a, V, and B.2--were isolated from a single human squamous cell carcinoma. Although all three cell lines can grow indefinitely in culture, they differ in a number of important transformation-related phenotypes. Only B.2 is strongly tumorigenic when injected into the flanks of nude mice, and only V can efficiently grow in semisolid media. The dominance of these traits was investigated by generating somatic cell hybrids among the three cell lines. F.2a was able to suppress the tumorigenicity of B.2 cells, whereas B.2 inhibited the capacity for anchorage-independent growth of V, the latter trait being a function of the ability of these epithelial cells to differentiate when deprived of support. The influence of exogenously added growth factors was also evaluated. This study indicates that the particular tumor we examined consisted of a heterogeneous population of cells with distinct growth and differentiation capacities.

Techniques used to freeze-dry "small doses" of thrombin.

In can be difficult to maintain the moisture level of small quantities of freeze-dried product contained in vials. We have observed that over a period of time a gradual increase in the residual moisture level in Thrombin vials containing small quantities of the freeze-dried product can sometimes be seen. No change in Thrombin potency has been observed. Investigations into the cause and resolution of this situation resulted in (1) modification of the freeze-drying cycle for vials containing small quantities of Thrombin and (2) incorporation of a special drying cycle for vial stoppers.

Measurement of bioreduction rates of cells with distinct responses to ionizing radiation and cisplatin.

A low frequency electron paramagnetic resonance (EPR) spectrometer has been used to measure the bioreduction rate of an exogenously added nitroxide free radical species. Measurements have been made in a well controlled, in vitro system using an X-ray and cisplatin sensitive Chinese hamster ovary (CHO) cell line, xrs-5, and partial revertants which display wild-type levels of sensitivity to X-rays but retain xrs-5 levels of cisplatin sensitivity. The xrs-5 cells reduce this radical species at a rate which is approx. 50% that of the wild-type CHO cell line, K1. The partial revertants maintain this defect in bioreduction despite their decrease in radiosensitivity. However, the bioreduction rate observed in these cells correlates with their sensitivity to the chemotherapeutic drug cisplatin. Low frequency EPR allows measurements and imaging of living tissue and may be of value as a predictive assay of human tumor response to chemotherapy.

Radioresistant derivatives of an X-ray-sensitive CHO cell line exhibit distinct patterns of sensitivity to DNA-damaging agents.

X-ray-resistant clones of the xrs-5 radiosensitive derivative of the CHO-K1 cell line were generated by transfecting cosmid library DNAs into the X-ray-sensitive cells. Transfectants were selected for both a dominant drug resistance marker present in the vector sequences and return to wild-type survival. Three cell lines were isolated which show X-ray survival characteristics similar to parental K1 cells. These revertant lines were examined for their cross-sensitivity to cis-diamminedichloroplatinum(II) (cisplatin) and bleomycin. Although these cell lines reverted with regard to X-ray sensitivity, they retained their sensitivity to cisplatin. Furthermore, changes in bleomycin and X-ray sensitivity did not correlate. There was a positive correlation between return to wild-type radiosensitivity and an increase in the rate of DNA double-strand break rejoining.

Detection of distinct transforming genes in X-ray induced tumors.

DNAs from mouse skin tumors (papillomas, squamous cell carcinomas, basal cell carcinomas and pilomatrixomas) initiated with X-irradiation and promoted with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) demonstrated dominant transforming activity by the production of transformed foci in the mouse recipient line, NIH3T3. Dominant transforming activity was not found in DNA isolated from normal mouse epidermis or from the corresponding liver. The NIH3T3 transformants induced with squamous cell carcinoma DNA grew in soft agar and formed tumors in nude mice. Southern blot analysis of primary NIH3T3 transformant DNAs carrying oncogenes from radiation-initiated squamous cell carcinomas indicated that the oncogenes responsible for the transformation of the recipient cells were not Ha-ras, Ki-ras or N-ras genes, nor were they erbB, B-lym, met, neu or raf. The data presented indicate that DNAs from radiation-initiated mouse skin tumors contain dominant transforming genes that are detectable by DNA-mediated gene transfer. The oncogene sequences activated in these radiation-initiated tumors are distinct non-ras transforming genes.

Activation of the cellular Harvey ras gene in mouse skin tumors initiated with urethane.

Mouse skin tumors, benign papillomas, and squamous cell carcinomas (SCCs) were initiated by a single topical application of urethane followed by repeated promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). Using the NIH 3T3 focus forming assay, dominant transforming activity was detected in DNA isolated from SCC samples. Rearranged and amplified copies of the c-Ha-ras gene were detected in NIH 3T3 transformant cell lines, indicating that an activated Ha-ras gene had been transferred to the NIH 3T3 recipient cells. Analysis of p21ras from the transformant cell lines suggested that the activating ras mutation was present in codon 61. Ultimately, the Ha-ras gene was shown to be activated by a specific A----T transversion at the second position of codon 61. This mutation was detected in both benign papillomas and SCCs, suggesting the activation occurred early in tumor development. The results demonstrate a highly consistent activation of the Ha-ras oncogene by a specific point mutation, suggesting a functional role for an activated ras gene in the initiation of mouse skin tumors by urethane.

X-ray and cis-diamminedichloroplatinum(II) cross-resistance in human tumor cell lines.

Clinical studies have suggested a close correlation between cis-diamminedichloroplatinum(II) (cisplatin) and radiation resistance. To determine whether this cross-resistance is due to an inherent cellular resistance to both agents, ten early passage human tumor cell lines were examined for their radiation and cisplatin sensitivity in vitro. Previous studies have suggested that these early passage tumor cell lines retain many of their in vivo characteristics and are therefore good models for tumor cells in vivo. Radioresistance was strongly associated with cisplatin resistance in these cell lines. Four of the cell lines examined were radioresistant, having Dos greater than 2.0 Gy. These four lines were also resistant to cisplatin, with the dose reducing survival to 10% greater than 1.29 microM. The remaining six cell lines had Dos ranging from 1.07 to 1.57 Gy of X-ray and doses reducing survival to 10% of less than 0.83 microM cisplatin. Because early passage human tumor cell lines were used, resistance or sensitivity to radiation and cisplatin most likely developed in vivo and was not due to selection in vitro. These results indicate that cross-resistance between cisplatin and radiation in vivo is probably due primarily to an inherent cellular resistance to these agents and not necessarily to the tumor microenvironment in situ.

Ionizing radiation enhances malignant progression of mouse skin tumors.

Chemical carcinogenesis in mouse skin has been divided into the process of initiation, promotion and progression. Recently we have shown that ionizing radiation acts as an initiator in this model system. In this paper we describe a three-stage experiment using ionizing radiation in the third stage of mouse skin carcinogenesis. CD-1 mice were initiated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) followed by biweekly promotion with 12-O-tetradecanoylphorbol-13-acetate (TPA). After 20 weeks of promotion, the animals were treated with either acetone, TPA (twice a week for 2 weeks) or eight fractions of 1 MeV electrons (1 Gy/fraction over a period of 10 days). The conversion of papillomas to squamous cell carcinomas was 80% for animals treated with ionizing radiation compared with 25% for tumor-bearing animals treated with TPA. Ionizing radiation increased the number of cumulative carcinomas per group. The lack of an increase in the number of cumulative papillomas per group due to ionizing radiation suggests that the dose and fractionation protocol used in this study enhanced the progression of pre-existing papillomas.

Ionizing radiation as an initiator in the mouse two-stage model of skin tumor formation.

The initiating potential of a range (7.5 to 22.5 Gy) of 4 MeV X rays was studied using the mouse skin two-stage model of carcinogenesis. A single dose of radiation was followed by 60 weeks of promotion with 12-O-tetradecanoyl phorbol-13-acetate (TPA) (8 nmol, two times per week). Since the proliferative state of a target cell population is known to influence carcinogen-induced tumorigenesis, we also investigated the effect of TPA on tumor incidence when applied as a single dose (17 nmol) 24 h prior to irradiation. Evidence presented here indicates that ionizing radiation can act as an initiator in this model system. All groups of animals that were promoted with TPA developed papillomas regardless of radiation treatment; however, only those groups of animals that received irradiation followed by TPA promotion developed squamous cell carcinomas. The incidence of nonepidermal tumors was similar between all radiation dose groups and was independent of TPA promotion. Our results also indicate that TPA pretreatment prior to irradiation results in an overall increase in the total tumor incidence, including both epidermal and nonepidermal tumors.

Production of DNA single strand breaks in rabbit renal tissue after exposure to 1,2-dichlorovinylcysteine.

S-(trans-1,2-dichlorovinyl)-L-cysteine (DCVC) is a recognized nephrotoxin. To investigate the genotoxic effects of DCVC on the kidney, DNA strand breaks were measured as an indicator of DCVC induced damage. To ascertain if bioactivation of DCVC occurred in the kidney, 3 experimental systems were used: in vivo; isolated perfused kidneys; and isolated proximal tubules of albino male rabbits. A dose-dependent increase in strand breaks in the kidney tubular DNA occurred after in vivo dosing with 5-100 mg/kg DCVC and after in vitro exposure to 10(-5)-10(-2) M DCVC. These results demonstrate the genotoxic effect of this compound on renal tissue.

Chronic toxicity of S-(trans-1,2-dichlorovinyl)-L-cysteine in mice.

S-(trans-1,2-Dichlorovinyl)-L-cysteine (DCVC) exposure causes acute renal tubular cytotoxicity. To further characterize the effects of DCVC, a chronic study was undertaken. Male Swiss-Webster mice received DCVC dissolved in their drinking water at 0.01, 0.05 and 0.1 mg ml-1. At 4, 8, 21 and 37 weeks, animals were terminated. Bladders, spleens, livers, kidneys and eyes were removed for histopathological examination. At 0.05 and 0.1 mg ml-1 DCVC, growth retardation was evident by 21 weeks. By 26 weeks, all animals in the 0.1 mg ml-1 group had developed cortical cataracts. Cytomegaly, nuclear hyperchromatism and multiple nucleoli were noted in the cells of the pars recta region of the kidney by 4 weeks and correlated to time and dose. At later time points, renal tubular atrophy and early interstitial fibrosis were evident. The epithelial cytological cellular abnormalities appear to be dose-related. Minor pathological changes were noted in the spleen, while there was no effect on the liver or bladder. Chronic ingestion of DCVC results in severe kidney injury.

In vivo and in vitro nephrotoxicity of the cysteine conjugate of hexachlorobutadiene.

Hexachlorobutadiene (HCBD), a renal toxin and carcinogen, is thought to require bioactivation to exert toxicity. The chemically synthesized cysteine conjugate of structurally similar halogenated hydrocarbons, trichloroethylene, chlorotrifluoroethylene, and chlorodifluoroethylene, have been shown to be nephrotoxic. Hence the cysteine conjugate of HCBD, S-pentachlorobuta-1,3-dienyl cysteine (PCBC), was assessed for potential nephrotoxicity. Active acid and base transport in isolated rabbit renal tubules was used to screen nephrotoxicity. A dose-dependent decrease in acid and base transport was observed after incubation with PCBC. At 10(-5) M PCBC transport was similar to that in controls, while at 10(-3) M PCBC completely inhibited active transport. In addition, in vivo exposure of Swiss-Webster male mice caused dose-dependent damage in the pars recta region of the proximal tubules (5-25 mg/kg ip). Genotoxicity in renal tissue was studied by using alkaline elution to detect DNA single-strand breaks and total cross-links. No DNA single-strand breaks were observed in isolated rabbit renal tubules after exposure to 10(-3) to 10(-5) M PCBC. However, at 10(-3) M PCBC there was some evidence of DNA cross-links. Therefore if cysteine conjugates of HCBD are formed in vivo, they could account for the toxicity observed with exposure to HCBD.