Fbxo45-mediated degradation of the tumor-suppressor Par-4 regulates cancer cell survival.

Prostate apoptosis response protein 4 (Par-4) also known as PRKC apoptosis WT1 regulator is a tumor suppressor that selectively induces apoptosis in cancer cells. However, its post-translational regulation by ubiquitin-mediated proteolysis and the cellular machinery that is responsible for its proteasomal degradation are unknown. Using immunopurification and an unbiased mass spectrometry-based approach, we show that Par-4 interacts with the SPRY-domain containing E3 ubiquitin ligase Fbxo45 through a short consensus sequence motif. Fbxo45 interacts with Par-4 in the cytoplasm and mediates its ubiquitylation and proteasomal degradation. Fbxo45 silencing results in stabilization of Par-4 with increased apoptosis. Importantly, a Par-4 mutant that is unable to bind Fbxo45 is stabilized and further enhances staurosporine-induced apoptosis. Co-expression of Fbxo45 with Par-4 protects cancer cells against Par-4-induced apoptosis. Our studies reveal that Fbxo45 is the substrate-receptor subunit of a functional E3 ligase for Par-4 that has a critical role in cancer cell survival.

MMSET stimulates myeloma cell growth through microRNA-mediated modulation of c-MYC.

Multiple myeloma (MM) represents the malignant proliferation of terminally differentiated B cells, which, in many cases, is associated with the maintenance of high levels of the oncoprotein c-MYC. Overexpression of the histone methyltransferase MMSET (WHSC1/NSD2), due to t(4;14) chromosomal translocation, promotes the proliferation of MM cells along with global changes in chromatin; nevertheless, the precise mechanisms by which MMSET stimulates neoplasia remain incompletely understood. We found that MMSET enhances the proliferation of MM cells by stimulating the expression of c-MYC at the post-transcriptional level. A microRNA (miRNA) profiling experiment in t(4;14) MM cells identified miR-126* as an MMSET-regulated miRNA predicted to target c-MYC mRNA. We show that miR-126* specifically targets the 3'-untranslated region (3'-UTR) of c-MYC, inhibiting its translation and leading to decreased c-MYC protein levels. Moreover, the expression of this miRNA was sufficient to decrease the proliferation rate of t(4;14) MM cells. Chromatin immunoprecipitation analysis showed that MMSET binds to the miR-126* promoter along with the KAP1 corepressor and histone deacetylases, and is associated with heterochromatic modifications, characterized by increased trimethylation of H3K9 and decreased H3 acetylation, leading to miR-126* repression. Collectively, this study shows a novel mechanism that leads to increased c-MYC levels and enhanced proliferation of t(4;14) MM, and potentially other cancers with high MMSET expression.

NPM-ALK signals through glycogen synthase kinase 3ß to promote oncogenesis.

Anaplastic large cell lymphoma (ALCL) is the most common type of pediatric peripheral T-cell lymphoma. In 70-80% of cases, the chromosomal aberration t(2;5)(p23;q35) results in the juxtaposition of anaplastic lymphoma kinase (ALK) with nucleophosmin (NPM) and the subsequent expression of the NPM-ALK fusion protein. NPM-ALK is a chimeric tyrosine kinase, which induces numerous signaling pathways that drive proliferation and abrogate apoptosis. However, the mechanisms that lead to activation of downstream growth regulatory molecules have not been completely elucidated. Using a mass spectrometry-based phosphoproteomic screen, we identified GSK3ß as a signaling mediator of NPM-ALK. Using a selective inhibitor of ALK, we demonstrated that the tyrosine kinase activity of ALK regulates the serine-9 phosphorylation of GSK3ß. Expression of NPM-ALK in 293T cells led to an increase of pS(9)-GSK3ß (glycogen synthase kinase 3 beta) compared with kinase-defective K210R mutant NPM-ALK, but did not affect total GSK3ß levels. Phosphorylation of pS(9)-GSK3ß by NPM-ALK was mediated by the PI3K/AKT signaling pathway. ALK inhibition resulted in degradation of GSK3ß substrates Mcl-1 and CDC25A, which was recovered upon chemical inhibition of the proteasome (MG132). Furthermore, the degradation of Mcl-1 was recoverable with inhibition of GSK3ß. ALK inhibition also resulted in decreased cell viability, which was rescued by GSK3ß inhibition. Furthermore, stable knockdown of GSK3ß conferred resistance to the growth inhibitory effects of ALK inhibition using viability and colony formation assays. pS(9)-GSK3ß and CDC25A were selectively expressed in neoplastic cells of ALK+ALCL tissue biopsies, and showed a significant correlation (P<0.001). Conversely, ALK-ALCL tissue biopsies did not show significant correlation of pS(9)-GSK3ß and CDC25A expression (P<0.2). Our results demonstrate that NPM-ALK regulates the phosphorylation of S(9)-GSK3ß by PI3K/AKT. The subsequent inhibition of GSK3ß activity results in accumulation of CDC25A and Mcl-1, which confers the advantage of growth and protection from apoptosis. These findings provide support for the role of GSK3ß as a mediator of NPM-ALK oncogenesis.

A model for melanosome biogenesis based on the purification and analysis of early melanosomes.

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.

Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase.

Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.

Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action.

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

An approach to proteomic analysis of human tumors.

A strategy for proteomic analysis of microdissected cells derived from human tumor specimens is described and demonstrated by using esophageal cancer as an example. Normal squamous epithelium and corresponding tumor cells from two patients were procured by laser-capture microdissection and studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Fifty thousand cells resolved approximately 675 distinct proteins (or isoforms) with molecular weights ranging between 10 and 200 kDa and isoelectric points of pH 3-10. Comparison of the microdissected protein profiles showed a high degree of similarity between the matched normal-tumor samples (98% identical). However, 17 proteins showed tumor-specific alterations, including 10 that were uniquely present in the tumors and seven that were observed only in the normal epithelium. Two of the altered proteins were characterized by mass spectrometry and immunoblot analysis and were identified as cytokeratin 1 and annexin I. Acquisition of 2D-PAGE protein profiles, visualization of disregulated proteins, and subsequent determination of the identity of selected proteins through high-sensitivity MS-MS microsequencing are possible from microdissected cell populations. These separation and analytical techniques are uniquely capable of detecting tumor-specific alterations. Continued refinement of techniques and methodologies to determine the abundance and status of proteins in vivo holds great promise for future study of normal cells and associated neoplasms. Mol.

Prostate secretory protein (PSP94) suppresses the growth of androgen-independent prostate cancer cell line (PC3) and xenografts by inducing apoptosis.

BACKGROUND: PSP94 (prostate secretory protein of 94 aa; also called PIP), one of the predominant proteins secreted into the seminal fluid, was proposed as an independent diagnostic/prognostic marker for prostate cancers. It was also shown to inhibit rat prostate cancer growth. In this study, we investigated the effect of purified PSP94 on the growth of androgen-independent human prostate cancer cells (PC3) and its potential mechanism of action. METHODS AND RESULTS: PSP94, in a dose- and time-dependent manner, inhibited the growth of PC3 cells. The protein demonstrated a stronger inhibitory effect on the colony-forming ability of PC3 cells in soft agar. A daily injection of PSP94 at 5 microg/kg/body weight resulted in a 50-60% inhibition in the growth of PC3 xenografts in athymic mice. PC3 cell growth inhibition by PSP94 resulted from cell death characteristic of morphological apoptosis, which was confirmed by dual fluorescence microscopy, electron microscopy, and DNA fragmentation assays. Mechanistic studies indicated that PSP94 enhanced the expression of proapoptotic protein Bax without affecting Bcl-2 levels. CONCLUSIONS: This study suggests that PSP94 may represent a novel, apoptosis-based, antitumor agent applicable to the treatment of hormone-refractory human prostate cancers.

Environmental estrogen stimulation of growth and estrogen receptor function in preneoplastic and cancerous human breast cell lines.

BACKGROUND: DDT and polychlorinated biphenyls (PCBs), which are widespread in the ecosystem, can mimic estrogen-mediated cell activities. Thus, they can potentially interfere with many physiologic processes. We compared the effects of organochlorines belonging to the DDT and PCB families, alone and in combination, for their ability to influence the estrogen receptor-mediated activities in preneoplastic breast epithelial cells and breast cancer cells. METHODS: Multiple assay systems requiring functional estrogen receptor were employed to test estrogen-like activity of organochlorine ligands. Two-sided statistical tests were used to compare the data. RESULTS: p,p'-DDT, the predominant form of DDT in the environment, is a more potent estrogen than o,p'-DDT (P<.001), although it is less effective than o,p'-DDT in inhibiting the binding of estradiol (natural estrogen) to estrogen receptor. Among the PCBs, Heptachlor is estrogenic (in transient reporter assays; P< or =.001), whereas Aroclor 1221 and Aroclor 1254, both individually and in combination, are only weakly estrogenic. CONCLUSION: p,p'-DDT is the most effective organochlorine in regulating estrogen receptor-mediated cellular responses. In estrogen receptor-positive breast cancer cells, p,p'-DDT evokes responses by itself and enhances the responses in collaboration with estradiol or o,p'-DDT.

Modulation of cisPlatin cytotoxicity by interleukin-1 alpha and resident tumor macrophages.

The modulation of cisPlatin cytotoxicity by interleukin-1 (IL-1 alpha) was studied in cultures of SCC-7 tumor cells with and without tumor macrophages to examine potential mechanisms for the synergistic antitumor activity of cisPlatin and IL-1 alpha in SCC-7 solid tumors. Neither IL-1 alpha nor tumor macrophages affected the survival of clonogenic tumor cells and IL-1 alpha had no direct effect on tumor cell growth in vitro. Macrophages had no direct effect on cisPlatin sensitivity (IC90 = 6.0 microM), but, the addition of IL-1 alpha (500-2000U/ml) to co-cultures of cisPlatin pretreated tumor cells and resident tumor macrophages increased cell killing (IC90 = 3.1 microM). Similar responses were seen in primary cultures treated with cisPlatin before IL-1 alpha. The modulation of cisPlatin cytotoxicity by IL-1 alpha exhibited a biphasic dose response that paralleled the IL-1 alpha dose dependent release of H2O2 by resident tumor macrophages. Further, IL-1 alpha modification of cisPlatin cytotoxicity was prompt and inhibited by catalase. CisPlatin and exogenous H2O2 (50 microM) produced more than additive SCC-7 clonogenic cell kill and hydroxyl radicals played an important role in the response. Interleukin-1 modulation of cisPlatin cytotoxicity was schedule dependent. IL-1 alpha treatment for 24 hrs, before cisPlatin, produced drug resistance (IC90 = 11.1 microM). Our study shows that IL-1 alpha can stimulate tumor macrophages to release pro-oxidants that modify cellular chemosensitivity in a schedule and dose dependent fashion. Our findings may also provide a mechanistic explantation for the synergistic antitumor activity of cisPlatin and IL-1 alpha in vivo.

Radioresistance in murine solid tumors induced by interleukin-1.

Interleukin-1 (IL-1) has radioprotective activity in hematopoietic lineages and in other normal cell renewal systems, but little is known about the effects of IL-1 alpha on the radiosensitivity of tumor cell populations. The present studies were conducted to investigate the effects of IL-1 alpha on the radiosensitivity of clonogenic cells in RIF-1 and SCC-7 tumors. Radioresistance was detected within 2-4 after administration of IL-1 alpha (0.5 micrograms/mouse, ip) and characterized by increases in D(o), Dq, alpha/beta and SF2. This radioresistance was similar to that seen in tumors rendered totally hypoxic before X irradiation. Tirapazamine, a hypoxic cell cytotoxin, and IL-1 alpha had synergistic schedule-dependent antitumor activity in vivo, suggesting that IL-1-induced radioresistance in vivo is due to hypoxia. Radioresistance induced by IL-1 alpha was transient, and the data suggested reoxygenation within 12 h. In vitro, IL-1 alpha had no direct effect on the radiosensitivity of SCC-7 cells in tissue culture under aerobic conditions. However, an increase in D(o), alpha/beta and SF2 was seen in clonogenic tumor cells from primary cultures treated with IL-1 alpha under aerobic conditions. Superoxide dismutase and catalase prevented the induction of radioresistance by IL-1 alpha in vitro, suggesting that oxidative responses from tumor macrophages after administration of IL-1 alpha may be responsible for induced radioresistance by IL-1 in vitro. Although oxidant stress induced by IL-1 and in vitro in our models, the mechanisms by which such responses modulate tumor radiosensitivity in vivo and in vitro are likely quite different.

The value of endorectal ultrasonography in the follow-up of intracavitary radiation treated early rectal cancer.

In selected patients with early rectal cancer, intracavitary radiation is a successful treatment. Endorectal ultrasound has proved an accurate method for staging and selecting such cancers for treatment. The value of endorectal ultrasound in the follow-up of patients with intracavitary radiation has not been previously assessed. Between 1989 and 1991, 30 patients treated at the Hamilton Regional Cancer Centre with intracavitary radiation were assessed by endorectal ultrasound. The mean age was 65 +/- 12 years with a range of 37-78 years. There were 17 males and 13 females. All patients were treated with curative intent. The dose of radiation administered was 8963 +/- 1506 cGy over 3.5 +/- 0.7 fractions. No patient received supplemental iridium implantation. Thirty-seven endorectal ultrasounds were carried out in 30 of the intracavitary radiation treated patients. Clinical findings (digital, sigmoidoscopic, and histology) were compared with the radiologist's interpretation of the endorectal ultrasounds. Using a 2 x 2 table accepting the clinical findings as the 'Gold Standard', the sensitivity of endorectal ultrasound was 71%, the specificity 61%, the positive predictive value 53%, the negative predictive value 78% with an overall accuracy of 75%. We conclude that endorectal ultrasound in the routine follow-up of patients treated with intracavitary radiotherapy for carcinoma of the rectum is questionable.

The influence of age on the management of anal cancer.

The standard treatment for anal cancer is combination chemo-radiotherapy. Management decisions such as radical chemotherapy, resective surgery for poor response or relapse are frequently modified by age-associated comorbid factors. Between 1980 and 1990, our regional cancer center serving a population of 1.8 million saw 78 patients with squamous carcinoma of the anus. We have compared patients who were younger than 65 years (n = 38) with those older than 65 years (n = 38). The mean +/- standard deviation age for the whole cohort was 65 +/- 12 years, with a ratio of 2 females to each male presenting. Fewer of the elderly age group had major surgery (26% vs. 42%) (p = 0.03), and fewer suffered no toxicity (42% vs. 26%) (p = 0.03). However, 61% of the under-65-year age group are alive disease-free vs. 26% of the elderly group (p = 0.03). Similarly, only 18% of the under-65-year group died with disease compared with 37% of the elderly group (b = 0.03). For the series as a whole, the crude mortality was 42%, with 27% dying of their disease. The stage distribution, and the amount of radiotherapy or chemotherapy administered was not age-specific, but younger patients had more surgery and suffered more toxicity, with a greater proportion remaining alive and disease-free, and fewer dying of their disease. These data suggest that a more aggressive multi-modality approach in the elderly may improve disease response and survival.

Synergistic antitumor activity of cisplatin and interleukin 1 in sensitive and resistant solid tumors.

The antitumor activity of cis-diamminedichloroplatinum(II) (cP) and human recombinant interleukin-1 alpha (IL-1 alpha) was studied in RIF-1 and SC VII solid tumor models and in a cP-resistant subline of RIF-1 designated RIF-R1cP. In RIF-1 tumors, clonogenic cell survival after cP plus IL-1 alpha combinations was highly schedule and IL-1 alpha dose dependent. More than additive clonogenic cell kill was seen when cP was given 6 h before, but not 8 h before or at 2-6 h after IL-1 alpha. Time course studies indicated that maximal clonogenic cell killing was achieved within 4-6 h after the cP plus IL-1 alpha combination, with little or no recovery for up to 24 h. In vivo dose-response studies indicated that cP plus IL-1 alpha combinations induced more clonogenic cell kill than cP alone in all three tumor models, and analysis by the median effect principle indicated highly synergistic antitumor activity. Dexamethasone but not indomethacin inhibited the synergistic interaction. IL-1 alpha had no effect on the cytotoxicity of cP in SCC VII cells in vitro, and neither in vitro hypoxia nor in vivo ischemia, induced by clamping tumor blood supply, significantly affected cP clonogenic cell killing. Increased clonogenic cell killing was seen, however, after removal of the clamp, implicating reperfusion events, such as oxyradical stress, as a potential mechanism for increased cP cytotoxicity in SCC VII solid tumors. The data from our model systems provide a rationale for additional work to define the mechanisms of the synergistic antitumor activity of the cP plus IL-1 alpha combination and indicate that IL-1 alpha might be a useful adjunct to increase the clinical efficacy of cP-containing strategies for both sensitive and cP-resistant cancers.

Randomized clinical trial to assess the effectiveness of breast irradiation following lumpectomy and axillary dissection for node-negative breast cancer.

BACKGROUND: Although the conservation management of breast cancer has become a routine method of treatment in most centers, there is still considerable controversy surrounding the ultimate minimum treatment required for node-negative breast cancer to achieve adequate local control. PURPOSE: Our purpose was to assess the value of breast irradiation in reducing breast relapse following conservation surgery for node-negative breast cancer. We attempted to define low-risk groups of women for breast and distant site relapse (i.e., recurrence outside the breast) who might be spared breast irradiation or adjuvant systemic therapy. METHODS: Eight hundred thirty-seven patients were randomly assigned to receive radiation therapy or no radiation therapy following lumpectomy and axillary dissection for node-negative breast cancer. RESULTS: Breast irradiation reduced relapse in the breast from 25.7% in the controls to 5.5% in the irradiated patients. There was no difference in survival between the two groups (median follow-up, 43 months). A low-risk group (less than 5% chance of relapse in the breast without irradiation) could not be defined. Tumor size (greater than 2 cm), age (less than 40 years), and poor nuclear grade were important predictors for breast relapse. Age (less than 50 years) and poor nuclear grade were important predictors for mortality. The presence of ductal carcinoma in situ did not predict breast relapse. CONCLUSIONS: Breast irradiation significantly reduces breast relapse, but it does not influence survival. Important predictors of breast relapse are age, tumor size, and nuclear grade, but not the presence of ductal carcinoma in situ. Age and, in particular, nuclear grade predict survival. IMPLICATIONS: Further follow-up may define an acceptable low-risk group for breast relapse. Until then, we recommend that all patients receive breast irradiation. Systemic adjuvant therapy should be considered for patients with poor nuclear grade tumors.

Methotrexate/fluorouracil scheduling influences normal tissue toxicity but not antitumor effects in patients with squamous cell head and neck cancer: results from a randomized trial.

To test the hypothesis that sequential scheduling of methotrexate (MTX) and fluorouracil (FU) produces a synergistic antitumor effect, we randomized 113 patients with recurrent or locally advanced squamous cell carcinoma of the head and neck to receive MTX-FU either 18 hours apart or simultaneously, with leucovorin rescue. There were 100 patients with locally advanced newly presenting disease and 13 patients with recurrence. Excessive toxicity was observed in the first 11 patients who received MTX 250 mg/m2 administered intravenously (IV) and leucovorin at 36 hours, therefore all subsequent patients received MTX 200 mg/m2 administered IV and leucovorin at 24 hours. FU 600 mg/m2 IV was administered to all patients, and treatment was given on days 1 and 8 of 21-day cycles. The treatment groups were well balanced for known prognostic variables. The response rate was 47.3% (26 of 55) for simultaneous v 44.8% (26 of 58) for sequential therapy. These results exclude a 20% difference in response rate favoring sequential therapy at P = .04. There was no observed difference in survival between the two treatment arms (P = .55) with a minimum follow-up of 8 months. Toxicity was greater in patients who received sequential therapy, and the difference was confined to the gastrointestinal (GI) tract. A comparison of the distribution in maximum Eastern Cooperative Oncology Group (ECOG) toxicity scores during chemotherapy for the two treatment groups showed greater stomatitis (P = .001), diarrhea (P = .04), and overall toxicity (P = .02) for sequential treatment without an observed difference in bone marrow toxicity. The results of this trial indicate that sequential MTX-FU is not superior to simultaneous therapy for the treatment of patients with head and neck cancer. Biochemical modulation of MTX-FU by drug scheduling may occur in vivo and may be organ specific.

Modulation of in vitro response to adriamycin by verapamil in murine P388 leukaemia, Ehrlich ascites carcinoma and sarcoma 180.

Experiments were carried out in vitro on adriamycin (ADR) accumulation and cytotoxicity alone and in combination with calcium channel antagonist, verapamil (VRP), in ascites murine tumour models of Ehrlich carcinoma (EAC), sarcoma 180 (S180) and P388 lymphocytic leukaemia (P388). The cytotoxicity was assayed as the inhibition of [3H]-thymidine incorporation into the cellular DNA. ADR, a broad-spectrum anticancer drug, at a concentration of 10 micrograms/ml showed a cytotoxic effect in the order S180 greater than P388 greater than EAC. VRP alone exhibited the DNA synthesis inhibiting activity. The enhanced DNA biosynthesis inhibition of ADR along with VRP was maximal in S180 and marginal in P388 and EAC. The ADR retention after 3 hr of incubation in these tumour models correlated with the cytotoxicity. VRP enhanced the accumulation of ADR in all these cell lines. The property of potentiation in the activity and accumulation of ADR in these tumours exposed to a non-toxic concentration of VRP can best be utilized in cancer chemotherapy where the massive cytotoxic therapy, with a single large dose of ADR, can be substituted with a low dose, along with this drug-response modulator, for better therapeutic results.

Restoration of drug sensitivity by nitroxazepine hydrochloride in P388 murine leukemia cells resistant to adriamycin.

Attempts were made to reverse the acquired resistance of P388 murine leukemia utilizing non-toxic concentration of nitroxazepine hydrochloride, an antidepressant. The effect of nitroxazepine hydrochloride on the intracellular accumulation of adriamycin and the inhibition of DNA synthesis were studied in these cells. The survival of mice bearing adriamycin-resistant P388 murine leukemia cells treated in vitro with nitroxazepine hydrochloride, adriamycin and a combination of the two drugs was also investigated. The results show that treatment of these cells with nitroxazepine hydrochloride significantly enhanced (1) the intracellular accumulation of adriamycin, (2) inhibition of DNA biosynthesis, and (3) the survival of mice transplanted with adriamycin-resistant P388 murine leukemia cells treated in vitro with a combination of nitroxazepine hydrochloride and adriamycin. The mechanism of restoration of drug sensitivity by nitroxazepine hydrochloride in adriamycin-resistant P388 cells could be due to an enhanced intracellular accumulation of adriamycin. The implications of the present investigations are promising, leaving hope for the utility of nitroxazepine hydrochloride in restoring drug sensitivity in adriamycin-resistant tumors.

Augmentation of hydroxyurea cytotoxicity by sintamil in human chronic myeloid leukemia cells.

The in vitro effect of sintamil, as a modulator alone and in combination with hydroxyurea (HU), on cytotoxicity was studied in 16 cases of human chronic myeloid leukemia (CML). We investigated the cytotoxicity of the drugs as a function of the exposure dose (HU, 10(-4) M; sintamil, 10 micrograms/ml) and the exposure time (1 h) to the agent. Cytotoxicity was evaluated as the inhibition of incorporation of [3H-methyl]thymidine in the nucleic acids of CML cells. Cytotoxicity of HU was greatly enhanced (P less than 0.001) by 1 h exposure of the CML cells to sintamil. The present data indicate that sintamil potentiates the cytotoxic activity of HU in CML cells.