H-ras-transformed NRK-52E renal epithelial cells have altered growth, morphology, and cytoskeletal structure that correlates with renal cell carcinoma in vivo.

We studied the effect of the ras oncogene on the growth kinetics, morphology, cytoskeletal structure, and tumorigenicity of the widely used NRK-52E rat kidney epithelial cell line and two H-ras oncogene-transformed cell lines, H/1.2-NRK-52E (H/1.2) and H/6.1-NRK-52E (H/6.1). Population doubling times of NRK-52E, H/1.2, and H/6.1 cells were 28, 26, and 24 h, respectively, with the transformed cells reaching higher saturation densities than the parent cells. NRK-52E cells had typical epithelial morphology with growth in colonies. H/1.2 and H/6.1 cell colonies were more closely packed, highly condensed, and had increased plasma membrane ruffling compared to parent cell colonies. NRK-52E cells showed microfilament, microtubule, and intermediate filament networks typical of epithelial cells, while H/1.2 and H/6.1 cells showed altered cytoskeleton architecture, with decreased stress fibers and increased microtubule and intermediate filament staining at the microtubule organizing center. H/1.2 and H/6.1 cells proliferated in an in vitro soft agar transformation assay, indicating anchorage-independence, and rapidly formed tumors in vivo with characteristics of renal cell carcinoma, including mixed populations of sarcomatoid, granular, and clear cells. H/6.1 cells consistently showed more extensive alterations of growth kinetics, morphology, and cytoskeleton than H/1.2 cells, and formed tumors of a more aggressive phenotype. These data suggest that analysis of renal cell characteristics in vitro may have potential in predicting tumor behavior in vivo, and significantly contribute to the utility of these cell lines as in vitro models for examining renal epithelial cell biology and the role of the ras proto-oncogene in signal transduction involving the cytoskeleton.

Use of long RT-PCR to characterize splice variant mRNAs.

Recent advances in long reverse transcription (RT)-PCR technology allow the copying of full-length coding regions of large mRNAs in one step. Using long RT-PCR, one can be certain that a given cDNA is derived from a single mRNA. In what to our knowledge is a novel application, we can isolate and characterize splice variants for any given mRNA in a systematic manner. We optimized long RT-PCR to copy the full-length coding region of human multidrug resistance (MDR1) mRNA or the major vault protein (MVP) mRNA in one step, so that only one full-length PCR product was synthesized in each case. Such stringent conditions are necessary to ensure that smaller than full-length products derived from total cell RNA are true splice variants. Twenty MDR1 double-stranded (ds) cDNAs, isolated from either the full-length or one prominent splice-variant DNA band, visualized on agarose gels, were cloned and sequenced. Two were full-length, wild-type in sequence as expected, and the rest were splice-variant mRNAs. Fourteen of the clones were identical and encoded a prominent splice-variant mRNA that can be detected in two tumor cell lines. This approach is shown to be generally applicable to the systematic analysis of splice-variant mRNAs derived from any gene.

Comparison of the antitumor activity of gemcitabine and ara-C in a panel of human breast, colon, lung and pancreatic xenograft models.

Gemcitabine is a new deoxycytidine analog that exhibits significant cytotoxicity against a variety of cultured murine and human tumor cells. The cytotoxic action of gemcitabine appears to be due to the inhibition of DNA synthesis by inhibition of ribonucleotide reductase and by competition with dCTP for incorporation into DNA. We have previously shown that gemcitabine, but not cytosine arabinoside (ara-C), has a broad spectrum of antitumor activity against 7 different types of murine solid tumors. The activity of gemcitabine was schedule dependent. To further characterize its activity, gemcitabine was tested against 12 human carcinoma xenografts. When given on an every 3 day x 4 schedule, the following percent inhibitions (at maximally tolerated doses [MTD]; MTD/2) in tumor growth were seen: MX-1 mammary (93%; 80%), CX-1 colon (92%; 82%), HC-1 colon (96%; 92%), GC3 colon (98%; 94%), VRC5 colon (99%; 100%), LX-1 lung (76%; 61%), CALU-6 lung (75%; 38%), NCI-H460 lung (45%; 46%), HS766T pancreatic (73%; not tested), PaCa-2 pancreatic (69%; 40%), PANC-1 pancreatic (70%; 60%), and BxPC-3 pancreatic (9%; 19%). In contrast, only the LX-1 lung carcinoma xenograft was responsive to ara-C treatment, which inhibited tumor growth by a marginal 62 percent. Thus, like its activity against murine solid tumors, gemcitabine has excellent antitumor activity against a broad spectrum of human solid tumors.

Raloxifene (LY156758) produces antimetastatic responses and extends survival in the PAIII rat prostatic adenocarcinoma model.

The benzothiophene antiestrogen, raloxifene (LY156758), has selective estrogen pharmacological antagonist activity in rats. The PAIII rat prostatic adenocarcinoma model was used to evaluate the effects of this agent on the lymphatic and pulmonary metastasis and survival in tumor-bearing male Lobund-Wistar (LW) rats. Raloxifene was inactive against colony formation of PAIII cells in vitro. Similarly, following subcutaneous (s.c.) implantation of 10(6) PAIII cells in the tail, s.c. administration of raloxifene (2.0, 10.0, or 20.0 mg/kg/day) for 30 days failed to demonstrate cytoreductive activity against primary tumor growth in the tail. However, in these same animals, raloxifene administration produced significant (P < 0.05) inhibition of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximal responses = 89% and 81% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by raloxifene treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (P < 0.05) reduced by raloxifene administration in a dose-related manner (maximal reduction = 97% from control values). In these animals, maximal regression of 20% for ventral prostate and 21% for seminal vesicle were also seen after raloxifene administration (P < 0.05 for both). Coadministration of E2B and raloxifene had no consistent antagonistic effect upon the antitumor responses produced by raloxifene. Raloxifene (40.0 mg/kg/day for 28 days) produced marked decreases in PAIII metastasis in the lymphatic and pulmonary components. Continued administration of the compound produced significant (P < 0.05) extension of survival of PAIII-bearing rats. Further studies are needed to define the maximal antitumor efficacy and the mechanism of action of raloxifene in urogenital solid tumor animal models. These data support the contention that raloxifene represents a class of active antimetastatic agents with potential efficacy in the treatment of hormone-insensitive human prostatic cancer.

Studies on the mechanism of sulofenur and LY295501 toxicity: effect on the regulation of cytosolic calcium in relation to cytotoxicity in normal and tumorigenic rat kidney cell lines.

Treatment of NRK-52E (normal) and H/1.2-NRK-52E (Harvey-ras transfected NRK-52E) rat kidney epithelial-like cells with two Eli Lilly antitumor compounds, sulofenur and LY295501 (15.6 microM-1000 microM) resulted in concentration- and time-dependent cell killing. Cytosolic Ca2+ became elevated in both cell lines in the presence of extracellular Ca2+ but only minimally in its absence. Both drugs were more toxic to the tumorigenic cells than to the normal cells, but LY295501 was significantly more toxic to both cells. The similarity in toxic response by both cell lines suggests a similar mechanism of toxic action for both drugs. Since LY295501 is highly toxic to tumorigenic cells but has a manageable dose-limiting toxicity it shows excellent potential for use in chemotherapy.

Development and characterization of normal colonic epithelial cell lines derived from normal mucosa of patients with colon cancer.

BACKGROUND: Researchers have tried for at least 20 years to develop a normal human colonic cell line suitable for in vitro studies of human colonic diseases. We report a breakthrough development of two normal colon-derived cell lines. They are designated NCM356 and NCM425. MATERIALS AND METHODS: The cells were collected from the histologically normal colonic margin of patients undergoing resection for colon adenocarcinomas and grown in culture. RESULTS: Since NCM356 and NCM425 have now been subcultured 22 and 19 times, each has undergone more than 40 population doublings. Neither cell line has shown evidence of terminal differentiation. Immunohistochemical characterization studies demonstrated that they are epithelial cells. They variably expressed subsets of other markers, including tumor markers, but did not grow in soft agar. NCM356 did not form tumors, whereas NCM425 was tumorigenic in immunodeficient mice. CONCLUSION: These two cell lines represent the first successful in vitro culture of human colonocytes derived from normal mucosa. NCM356 is closer to normal, but seems to represent an early stage of cell transformation, possibly correlated with immortalization. In contrast, in vitro culture of the NCM425 cell line appears to have selected for later progression to malignancy. These lines are important resources for studying colon cancer and the physiology of intestinal cells.

Evaluation of new anticancer agents against the MIA PaCa-2 and PANC-1 human pancreatic carcinoma xenografts.

Human pancreatic carcinoma xenograft models were developed from established MIA PaCa-2 and PANC-1 cell lines (ATCC, Rockville, MD). Tumors were maintained by serial trocar implantation in CD1 nu/nu mice, and attempts were made to test all drugs under optimal schedules at maximum tolerated doses. In both models, adriamycin, cisplatin, and 5-fluorouracil were inactive (< 60% inhibition of tumor weight), whereas gemcitabine (LY188011] produced modest activity (69% inhibition in MIA PaCa-2 and 76% inhibition in PANC-1. Major differences in tumor sensitivity were noted with diarylsulfonylureas (DSU) and taxol. The DSU (Sulofenur [LY186641] and LY295501) produced complete inhibition in the MIA PaCa-2 xenograft, but were inactive in PANC-1. Conversely, taxol produced 80% inhibition of PANC-1 tumor growth, but was inactive against MIA PaCa-2. In general, in vivo antitumor activity roughly correlated with in vitro tumor cytotoxicity with the exception of DSU. We have previously shown that DSU are extensively bound to albumin and that in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity. The MIA PaCa-2 and PANC-1 xenograft models may be useful for selecting potential candidates for therapy of human pancreatic cancer.

Comparative antitumor effects of hormonal ablation, estrogen agonist, estrogen cytotoxic derivative, and antiestrogen in the PAIII rat prostatic adenocarcinoma.

The effects of hormonal ablation, estrogen, estrogen-derived cytotoxic agent, and estrogen antagonist therapies used clinically were evaluated on in vitro colony formation, in vivo growth, and lymphatic and pulmonary metastasis of the PAIII tumor. Ventral prostatic and seminal vesicle weights were evaluated in the same animals to assess androgen-related responses. Estradiol, estramustine phosphate, and testosterone had no effects on PAIII colony formation in vitro. Castration, hypophysectomy, estradiol benzoate, and estramustine phosphate treatment of PAIII-bearing Lobund Wistar rats produced significant (P less than 0.05) regression of male accessory sex organs. Of these treatments, only hypophysectomy had significant (P less than 0.05) inhibitory effects on primary PAIII growth and lymphatic and pulmonary metastasis. LY117018 [6-hydroxy-2-(p-hydroxyphenyl)benzo(b)thien-3-yl-p-2-(l-pyrrolidin yl)ethoxy phenyl ketone] has antiestrogenic activity but produces no significant agonist responses. LY117018 had no effect upon PAIII colony formation in vitro. Following s.c. implantation of PAIII cells, LY117018 (2.0, 10.0, or 20.0 mg/kg s.c.) had no effect on primary tumor growth in the tail. In vitro LY117018 administration produced marked antimetastatic effects. In a dose-dependent manner, LY117018 inhibited PAIII metastasis to the gluteal (97%) and iliac lymph nodes (88%) (P less than 0.05 for both). LY117018 also maximally inhibited pulmonary metastasis by 86% (P less than 0.05). Maximal regression of 42% for ventral prostatic and 35% for seminal vesicle weights were also seen after LY117018 administration (P less than 0.05 for both). Co-administration of estradiol benzoate had no antagonistic effect upon the antitumor responses produced by LY117018. The mechanism of action of LY117018 is not known. The failure of estradiol benzoate to affect PAIII growth and metastasis supports the contention that the responses to LY117018 are not attributable to simple antagonism of estrogen action. LY117018 may be exerting its antitumor effects through autocrine, paracrine, or endocrine mechanisms. LY117018 represents a class of agents with potential utility in treating metastatic cancer of the prostate.

Inhibition of PAIII rat prostatic adenocarcinoma growth and metastasis by a new diarylsulfonylurea antitumor agent, LY181984.

LY181984 is a compound in a series of orally active diarylsulfonylureas with broad spectrum in vivo activity against syngeneic rodent and human xenograft tumor models. The PAIII rat prostatic adenocarcinoma model was used to evaluate the effects of this antitumor agent on the lymphatic and pulmonary metastasis of the tumor in male Lobund Wistar rats. LY181984 was inactive against the proliferation of PAIII cells in vitro. Following subcutaneous implantation of 10(6) PAIII cells in the tail, oral administration of LY181984 (25.0, 50.0, or 100.0 mg./kg./day) for 30 days had no significant effects on body weight gain. LY181984 treatment produced significant (p less than 0.05) dose-dependent inhibition of primary tumor growth in the tail (max. inhibition = 46% from untreated control levels). In these same animals, LY181984 administration produced significant (p less than 0.05) dose-dependent inhibiton of PAIII metastasis from the primary tumor in the tail to the gluteal and iliac lymph nodes (maximal responses = 79% and 80% from control values, respectively). PAIII metastasis to the lungs was significantly inhibited by oral LY181984 treatment. Numbers of pulmonary foci in PAIII-bearing rats were significantly (p less than 0.05) reduced by LY181984 administration in a dose-dependent manner (maximal reduction = 78% from control values). While the non-toxic doses (less than 100.0 mg./kg./day for 28 days) of LY181984 produced marked decreases in tumor growth and metastasis, administration of the compound had no effect on the survival of PAIII-bearing rats. These data support the contention that LY181984 represents a new class of orally active antitumor and antimetastatic agents with potential efficacy in the treatment of hormone-insensitive prostatic cancer. Further studies are needed to define maximal efficacy of LY181984 and other sulfonylurea agents in urogenital solid tumor animal models.

Drug treatments for metastasis of the Lewis lung carcinoma: lack of correlation between inhibition of lung metastasis and survival.

The abilities of the Eli Lilly compounds LY150310, LY189332, and LY135305 to inhibit spontaneous metastasis and to increase animal survival were evaluated. These compounds represent widely varied structures and were evaluated because they have been found to inhibit thromboxane synthetase, cyclooxygenase, and thrombin activation, respectively. These biochemical processes have been proposed in the literature as targets for antimetastatic drugs. The purpose of this investigation was twofold: (a) to compare the antimetastatic activities of the Eli Lilly compounds to those of the reference antimetastatic compounds nafazatrom and RA233, and (b) to examine the correlation between inhibition of spontaneous lung metastasis and survival. Spontaneous metastasis of the Lewis lung carcinoma was used to evaluate the antimetastatic activity of the compounds. In this model 5 x 10(5) tumor cells were implanted into the gastrocnemius muscle, the primary tumor was resected on Day 14, and metastatic lung lesions were counted on Day 25. Compounds were administered every 12 h on Days 5 through 19. Nafazatrom, LY150310, LY189332, and LY135305 were found to inhibit spontaneous lung metastasis in a dose-dependent manner. The ED50 values for the respective inhibitions with these compounds were 50, 0.5, 2, and 0.35 mg/kg/day; the respective therapeutic indexes (LD50/ED50) were 7, 180, 255, and 511. To evaluate the effect of nafazatrom, LY150310, LY189332, and LY135305 on animal survival, the compounds were given at maximally antimetastatic doses of 200, 60, 20, and 6 mg/kg/day, respectively. Two dosing schedules were used: (a) on Days 5 through 19 and (b) on Day 5 until death. Neither the median survival times nor the numbers of long-term survivors were significantly changed with any of the compounds at any dosing schedule. RA233, given to a maximally tolerated dose of 200 mg/kg/day on Day 5 until death, did not inhibit lung metastasis and did not increase median survival time. Postmortem examination of animals dosed with nafazatrom, LY150310, LY189332, and LY135305 showed complete inhibition in lung lesions and the appearance of lesions in the liver, kidney, spleen, and brain. The results of this investigation show that the effect a compound has on the number of metastatic lesions in a target organ may not be predictive of its effect on survival. To successfully translate laboratory data into the clinic, survival should be considered as a predictor of a compound's potential clinical utility.

Correlation of the in vivo anticoagulant, antithrombotic, and antimetastatic efficacy of warfarin in the rat.

Fibrin formation has been hypothesized to be an element of the metastatic process in cancer, and pharmacological interference with such fibrin formation has been proposed as a means of antimetastatic therapy. We have tested this hypothesis through an in vivo study of warfarin in two independent rat disease models--a model of chemical-injury-induced arterial thrombosis, and a model of spontaneous metastasis. We found 0.50 mg/kg-day warfarin to be uniformly lethal after two weeks treatment. The chronic dose of 0.25 mg/kg-day was non-toxic and produced effective anticoagulation and marked antithrombotic and antimetastatic activity. The 0.125 mg/kg-day dose produced a reduction in factor IIc (50%) and factor VIIc (70%), and resulted in statistically significant antithrombotic and antimetastatic activity. The 0.0625 mg/kg-day dose failed to reduce the vitamin K-dependent clotting factors, and failed to produce any antithrombotic or antimetastatic effects. The substantial correlation (very similar dose-response effects) among the anticoagulant, antithrombotic and antimetastatic efficacies of warfarin in the rat suggests that anticoagulation provides the pharmacological mechanism underlying both the antithrombotic and the antimetastatic effects. The poor therapeutic index we observed in the rat may be the attribute which limits the efficacy of warfarin in the treatment of human cancer.

Inhibitory effect of warfarin on the metastasis of the PAIII prostatic adenocarcinoma in the rat.

The PAIII rodent metastatic prostatic adenocarcinoma model was employed to evaluate the effects of dietary warfarin, a prototypic antagonist of thrombin generation on the lymphatic and pulmonary metastases of the tumor from the tail site of subcutaneous transplantation in male Lobund Wistar (LW) rats. In addition, the anticoagulant effects of warfarin were determined in the same animals. Warfarin, administered in the diet at concentrations equivalent to 0.063, 0.125 or 0.250 mg./kg. b.w. for 30 days had no effect on final body weight, gluteal or iliac lymph node weights. Significant (p less than 0.05) dose-dependent extensions of whole blood prothrombin (WBPT), activated partial thromboplastin (WBAPTT) and clotting times (WBCT) over control values were observed with warfarin treatment. Preliminary studies demonstrated that the 0.500 mg./kg. dose produced 50 per cent mortality at +14 days. Warfarin produced significant (p less than 0.05) dose-dependent decreases in the number of PAIII pulmonary metastases as indicated by reductions in dry lung weights and lung colony numbers when compared to untreated tumor-bearing controls. While the therapeutic index of warfarin is a limiting factor in clinical use as an antimetastatic agent, these results suggest that compounds capable of altering hemostatic mechanisms may be potential inhibitors of tumor metastasis. The PAIII prostatic adenocarcinoma model may be a useful system to quantitatively evaluate potential antimetastatic and cytotoxic agents.

Metastatic spread of the PAIII prostatic adenocarcinoma after implantation in the tail of the rat.

The spontaneous metastatic spread of a suspension of PAIII prostatic adenocarcinoma cells from the tail site of implantation was analyzed over a period of 5 weeks in male Lobund-Wistar (LW) rats. Following subcutaneous injection of the PAIII cells, the tumor metastasized through the primary lymphatic drainage. PAIII microfoci were evident in the gluteal and iliac lymph nodes prior to colonization of the lungs. Growth of the primary tumor was evidenced by significant weight differences of the tails of PAIII-bearing and control rats 1 week after tumor implantation. Time-dependent sequential spread of the adenocarcinoma was quantitated. Significant differences were noted between PAIII-bearing and control animals with respect to the gluteal lymph node weights (+2 weeks), iliac lymph node weights (+3 weeks), dry lung weights, and lung colony numbers (+4 weeks) after tumor implantation. During the course of these studies, the whole blood prothrombin, activated partial thromboplastin, and recalcification times for the PAIII-bearing animals were similar to those of the control group. These findings indicate that there were no gross changes in systemic blood coagulation accompanying the metastasis of PAIII cells from the primary tumor. The tumor in LW rats produced a consistent pattern of growth and metastasis that is suitable for quantitation. The PAIII prostatic adenocarcinoma is a sensitive and reproducible system that may be useful to evaluate potential antimetastatic and cytotoxic agents for the treatment of hormone-insensitive prostatic cancer.