Rodent Tumor Cell Research Articles

Intratumoral injection of an adenovirus expressing interleukin 2 induces regression and immunity in a murine breast cancer model.

Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

Gp330 on type II pneumocytes mediates endocytosis leading to degradation of pro-urokinase, plasminogen activator inhibitor-1 and urokinase-plasminogen activator inhibitor-1 complex.

Glycoprotein 330 (gp330) is a member of a family of receptors related to the low density lipoprotein receptor (LDLR). Although several ligands have been shown to bind gp330 in solid-phase assays, the ability of gp330 to mediate ligand endocytosis has not been demonstrated. To develop a cellular model for gp330 function we screened a variety of cultured cell lines and identified several that expressed this protein, including immortalized rat type II pneumocytes and a human and two rodent tumor cell lines. Using type II pneumocytes, endocytosis of a previously described gp330 ligand, urokinase (uPA) complexed with plasminogen activator inhibitor-1 (uPA:PAI-1) and two new ligands, PAI-1 and pro-uPA, was demonstrated. RAP, the 39 kDa receptor-associated protein known to antagonize ligand binding to gp330 in solid-phase binding assays, completely inhibited both internalization and degradation of the radiolabeled ligands by type II pneumocytes. This suggested that the clearance of these ligands was dependent on either gp330 or the LDLR-related protein (LRP), which shares several ligand-binding characteristics with gp330. By using polyclonal antibodies to gp330, the cellular internalization and degradation of the ligands were inhibited by 30-50%; remaining ligand internalization and degradation activity could be partially inhibited by polyclonal antibodies against LRP. These findings indicate that gp330, like other LDLR family members, mediates endocytosis of its ligands. In addition, gp330 acts in concert with LRP in type II pneumocytes to mediate clearance of a variety of proteins involved in plasminogen activation, including uPA:PAI-1 complexes PAI-1 and pro-uPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemosensitization of CCNU in KHT murine tumor cells in vivo and in vitro by the agent RB 6145 and its isomer PD 144872

The cytotoxicity and chemosensitizing potential of the nitrohetercyclic agent RB 6145 and its R enantiomer PD 144872 were determined in rodent tumor cells grown in tissue culture or as solid tumors. Using a clonogenic cell survival assay the degree of selective cytotoxicity of these bioreductive drugs was first determined in KHT/iv cells. Cells treated under hypoxic conditions were observed to be ~50–80-fold more susceptible to the action of RB 6145 or PD 144872 than were cells exposed under air. To assess the in vitro chemosensitizing potential of RB 6145 and PD 144872, doses of these agents which led to survival values between 0.5 and 1.0 under hypoxic conditions were administered, and were then combined concomitantly with variable doses of the nitrosourea CCNU. Exposure to the nitrosourea was for 1 h. The results showed that inclusion of either sensitizer enhanced the cell killing of the chemotherapeutic agent 2.4-2.6-fold. Subsequent experiments evaluated the therapeutic benefit of combining these bioreductive agents with CCNU in KHT sarcoma-bearing C3H/HeJ mice. When given at times ranging from 90 min before to 60 min after CCNU exposure, these bioreductive drugs increased the tumoricidal activity of the chemotherapeutic agent. Complete dose response curves combining RB 6145 and PD 144872 and a range of CCNU doses also were evaluated. The sensitizers (0.75 mmol/kg) were administered 30 min prior to the chemotherapeutic agent and survival of clonogenic tumor cells 22–24 h after treatment was used to assay tumor response. Normal tissue toxicity under similar treatment conditions was assessed using a bone marrow stem cell (CFU-GM) assay. When combined with CCNU, the bioreductive agents increased tumor cell kill by a factor of ~1.7. Bone marrow toxicity was not, however, enhanced by the addition of either agent to the CCNU treatment. These data suggest that further consideration should be given to the clinical application of these bioreductive agents.

In vitro cytotoxicity and chemosensitizing activity of the dual function nitroimidazole RB 6145

Purpose : To determine the cytotoxicity and chemosensitizing potential of RB 6145 in mouse KHT/iv and human A549 tumor cells. Methods and Materials : RSU 1069, the lead compound in a series of nitroimidazoles containing an alkylating aziridine function, has been shown to possess a high degree of selective cytotoxicity for hypoxic cells in addition to being a potent sensitizer of radiation and chemotherapy. Unfortunately, preliminary clinical studies have revealed a dose-limiting gastrointestinal toxicity for RSU 1069. Recently RB 6145, the ring-opened analogue of RSU 1069, has been found to be less emetic than RSU 1069. In the present studies, we assessed both the differential hypoxic cell cytotoxicity of RB 6145 and its chemosensitizing potential when combined concomitantly with variable doses of the activated form of cyclophosphamide (4-hydroperoxy-cyclophosphamide, 4-OOH-CP) or the nitrosourea CCNU. Results : As we had observed previously for RSU 1069, RB 6145 was found to be less cytotoxic to human than rodent tumor cells. In addition, the degree of selective cytotoxicity toward hypoxic cells was (a) less in A549 than in KHT/iv cells (factor of 9 vs. 80) but (b) comparable to that seen with RSU 1069. For both cell lines, inclusion of the sensitizer enhanced the cell killing of the chemotherapeutic agent 4-OOH-CP by a factor of ∼ 1.5–1.7-fold. When combined with CCNU, RB 6145 increased the killing of A549 cells ∼ 1.8-fold. Similar hypoxic cell preferential cytotoxicity and enhancement in anti-tumor treatment efficacy were seen when A549 cells were exposed to the R enantiomer of RB 6145 (PD 144872) either alone or in combination with CCNU. Conclusion : These data support the notion that further consideration should be given to the clinical application of these Bioreductive agents.

Identification of partial complementary DNA clones encoding a 59-kd protein with characteristics of a unique oncofetal antigen.

Oncofetal antigens (OFAs) are conserved tumor-associated autoantigens or transplantation antigens present on the surface of all major classes of rodent and human tumors and on midgestational fetal cells but not on normal neonatal or adult human and rodent tissues. A syngeneically derived monoclonal antibody, MAb-115, recognizes murine OFAs of 44 and 200 kd in molecular mass. Our goal was to clone and characterize the complementary DNAs (cDNAs) that encode these murine OFAs. Rabbit antiserum raised against purified 44-kd OFA glycoprotein was used to screen a mouse embryo cDNA-lambda phage expression library. Recombinant phage clones positive for the expression of OFAs were detected by immunohistochemical staining, then isolated and plaque purified. The presence of an OFA-encoding sequence in the recombinant phage was confirmed by specific reaction of the expressed protein with MAb-115. Recombinant fusion protein was purified from the extracts of corresponding lysogens. Rabbit antiserum against purified recombinant fusion protein was raised, and the capacity of this antiserum to detect the expression of OFA on rodent tumor and fetal cells was determined by flow cytometry. In addition, immunoreactivity of tumor bearer and hyperimmune murine sera to bacterially expressed recombinant OFA protein was evaluated by enzyme-linked immunosorbent assay. The OFA-expressing insert DNA from plaque-purified lambda clones was subcloned into phagemid vectors for sequencing analysis. Antiserum derived against the isolated recombinant mouse embryo polypeptide mimicked MAb-115 in its specific binding to all OFA-positive rodent tumor and fetal cell lines tested and likewise did not show reactivity to normal adult tissues. This antiserum specifically recognized the native 44- and 200-kd OFAs in extracts of murine lymphocytic lymphoma. Furthermore, sera of tumor-bearing mice or mice immunized with purified OFA or intact, irradiated OFA-positive lymphocytic lymphoma cells also reacted with the recombinant fusion protein. The characterization of the isolated clone included nucleotide sequence information followed by analysis of the deduced primary structure of the protein. These data suggest that the isolated cDNA clones encode a distinct gene product which is widely expressed on the surface of tumor and fetal cells and represents the first characterized sequence of a true OFA. The availability of this cDNA, encoding a protein expressed only on tumor and fetal cells, provides a direct means to assess biological characteristics of malignant tissue which can be assayed by biochemical, histochemical, and molecular methods.

Factors affecting sensitivity to EO9 in rodent and human tumour cells in vitro: DT-diaphorase activity and hypoxia

Twenty-three human tumour cell lines (lung, breast, and colon) and eight rodent cell lines were evaluated for their sensitivity to the quinone-based anticancer drug EO9 [3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H indole-4,7-dione)prop-β-en-α-o1]. Sensitivity was compared with the intracellular levels of DT-diaphorase, and cell lines showing highest enzyme activity tended to be the most sensitive to EO9. The role of DT-diaphorase in determining drug sensitivity was confirmed by using the enzyme inhibitor dicoumarol, which protects cells containing high levels of DT-diaphorase from the cytotoxic action of EO9. Hypoxia increased the cytotoxicity of cells containing low but not high levels of DT-diaphorase, implying that both 1- and 2-electron reductive activation processes can be important for expression of EO9 toxicity. It is concluded that EO9 is a potentially useful agent in the enzyme directed approach to the use of bioreductive drugs in cancer therapy.

Differential expression of non-allelic insulin genes in rodent islet tumour cells

We have compared the expression patterns of the non-allelic insulin 1 and 2 genes during prolonged in-vitro culture of the mouse islet cell line beta-TC3, where transformation by the SV40 T oncoprotein is targeted to the differentiated beta-cell phenotype, and the rat islet cell line NHI-6F, in which the beta-cell phenotype is induced by transient in-vivo passage. The NHI-6F clone carries, in addition, a single copy of a transfected silent human insulin gene which contains 3 kb of regulatory sequences known to confer beta-cell-specific expression. Insulin gene expression was measured by an assay based on a reverse transcription-polymerase chain reaction, to determine whether the ancestral rodent insulin 2 genes (and the human homologue in the NHI-6F cells) are regulated differently from the duplicated rat and mouse insulin 1 genes. We have shown that activation of insulin gene expression in the NHI-6F cells includes transcriptional activation of all three genes, but that extended propagation of tumour cells in vitro leads to a selective and equal decline in the quantities of transcripts from the rat 2 and human genes relative to transcripts from the rat 1 gene. In the later passages, insulin transcripts were derived almost exclusively from the rat 1 gene. In early in-vitro passages of the mouse endocrine cell line beta-TC3, the expression pattern of the mouse 1 and 2 insulin genes resembled that seen in isolated mouse islets. After more than 45 in-vitro passages, expression of the duplicated mouse 1 gene decreased tenfold when compared with the ancestral mouse 2 gene. As previously shown for NHI-6F cells, the differential expression of non-allelic insulin genes in the beta-TC3 line was also clearly evident at the cellular level, where a subpopulation of cells selectively expressed readily detectable levels of mouse C-peptide 2 immunoreactivity while devoid of C-peptide 1. Our results suggest that the maintenance of insulin gene expression in rodent tumour cells is influenced by enhancer sequences which are not shared by the ancestral and duplicated insulin genes, and that either species-specific conditions or transformation-related differences exist between the rat and mouse cell lines that govern which gene remains active during prolonged in-vitro propagation.

Comparison of the potential therapeutic effects of interleukin 2 or interleukin 4 secretion by a single tumour.

Engineering of a variety of rodent tumour cells to secrete either interleukin 2 (IL-2), or interleukin 4 (IL-4), has been demonstrated to reduce their tumorigenicity. However the mechanisms of action of secreted IL-2 and IL-4 have not been compared in a single rodent tumour. Here we demonstrate that the weakly immunogenic murine fibrosarcoma FS29 had reduced growth rate and in some cases was rejected by syngeneic animals, when modified to secrete either IL-2 or IL-4, but not IL-5. Immunohistochemical analysis of tumour nodules undergoing regression showed stimulation of a largely lymphocytic infiltrate by IL-2 and a macrophage and granulocyte infiltrate, with a small number of lymphocytes by IL-4. Indeed, secretion of low levels of IL-2 and IL-4 in combination resulted in optimal rejection, suggesting that the two cytokines might mobilise different and complementary effector cell mechanisms. Both IL-2 and IL-4-secreting cells failed to induce the rejection of admixed, unmodified FS29 cells. The loss of cytokine secreting cells from such admixtures occurred more rapidly for IL-2-secreting cells. Injection of IL-4-secreting, but not IL-2-secreting FS29 cells could protect mice from a delayed challenge with unmodified FS29 cells. These data suggest that IL-4 secretion stimulates the better long-term host anti-tumour response.

Cellular stress- and transformation-associated cell surface antigens expressed on human and rodent tumor cells.

Stress‐induced proteins may have significant roles in anti‐tumor resistance. To clarify the immunobiological roles of these proteins, we first developed monoclonal antibody (mAb) H1A that detects the HeLa cell‐surface antigens whose expression was enhanced by treatment of the cells with physico‐chemical stressors, such as heat, H2O2 and tumor necrosis factor. H1A (IgM) detects several molecules with mol. wt. 30, 43, 75, 90, 100, 120 and 150 kDa in Western blot analysis of HeLa cell lysates. Although the antigen was constitutively expressed on the HeLa cell surface, the cell‐surface expression of H1A‐defined antigen was rapidly enhanced (within 1 h) after heat treatment of HeLa cells. H1A antigens were also transformation‐associated, since 1) the activated oncogene‐transformed fibroblasts expressed the antigens, but parental nontransformed cells did not, and 2) certain human neoplastic but not normal cells strongly expressed the antigens. Furthermore, H1A mAb also partly blocked the cytotoxicity of purified protein derivatives‐stimulated human T cell receptor γδ‐type T cells towards HeLa cells. Taken together, these data indicate that H1A‐defined stress‐inducible proteins may play a vital role in anti‐tumor resistance by cytotoxic T cells.

Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo

1. Vascular permeability factor (VPF) is a protein secreted from a variety of human and rodent tumour and normal tissue cells. In addition to mediating angiogenesis and endothelial cell growth, VPF has been reported to be a potent mediator of increased microvascular permeability in vivo. In this study we have investigated these permeability changes in vivo using a quantitative model of local plasma leakage in rabbit skin. 2. Our results reveal that VPF is a potent mediator of plasma leakage which, in the rabbit, depends on a synergistic interaction with arteriolar vasodilators such as prostaglandin E2. The requirement for an exogenous vasodilator further suggest that VPF does not act to increase blood flow in this model. 3. We show that this response does not require the presence of circulating neutrophils and in this respect is similar to direct-action permeability increasing mediators such as histamine and bradykinin. Similarly, the time course of plasma leakage induced by VPF resembles that of direct-action mediators, where the greatest response occurs over the first 30 min. In contrast, the neutrophil-dependent plasma leakage induced by the active component of zymosan-activated plasma, C5ades arg, was maintained at a similar level over 2.5 h. 4. Further, using mediator antagonists and enzyme inhibitors we demonstrate that the mechanism of action of VPF is not via activation of histamine, kinin, or platelet-activating factor pathways.

HUMAN TUMOR-CELL GROWTH MODULATORY EFFECTS OF THE AEBS/HIC-BINDING DRUGS USED AS SINGLE AGENTS AND IN COMBINATION WITH A NOVEL AMPHIBIAN OOCYTE RNASE

A novel amphibian oocyte RNase, ONCONASE(R) (ONC), has been previously shown to have synergistic tumor cell growth inhibitory activity when combined with tamoxifen (TMX) and/or trifluoperazine (TFP) in human ASPC-1 pancreatic and A-549 lung carcinoma cells, respectively. It has recently been reported that several drugs known to bind to the intracellular antiestrogen binding site (AEBS)/histamine (H(IC)) receptors, including tricyclic (amitriptyline, AMT) and non-tricyclic (fluoxetine, FLX) antidepressants, TMX, phenothiazines and the prototype H(IC)-binder DPPE, can stimulate the in vitro and in vivo growth of rodent tumor cells, while having a normal cell growth inhibitory activity, as reflected by the inhibition of the DNA synthesis. It has been presently shown that while at the clinically relevant concentrations some of these H(IC)-binding drugs mildly stimulated (up to 15%) the cell growth in the human lines studied when used as single agents, in most instances this stimulation did not exceed 10% above the control values. When used in combination with ONC, neither of these H(IC)-binding drugs demonstrated any apparent synergistic activity as judged from the ED50 values. However, the combinations of DPPE+TFP and AMT+TFP, in both the ASPC-1 and COLO 320DM lines, demonstrated a significant cell growth inhibition, while there was no difference between the effects of AMT alone and the AMT+TFP combination in the U87MG line. The most effective cell growth inhibition was obtained when ONC was combined with DPPE+TFP and/or AMT+TFP, as reflected by the significantly decreased ED50 values.

The characteristic which makes the cell-coded HSV-inducible U90 distinctive in transformed cells is its greatly increased half-life.

A set of polypeptides detected in transformed cells of M(r) values 90,000 (a doublet) 40,000 and 32,000 that are recognised by immunoprecipitation of L-[35S]methionine-labelled tumour cell lysates, with sera from tumour-bearing rats and with antisera raised against herpes simplex virus type 2 (HSV-2)-infected cells, were previously reported (Macnab et al., 1985, 1992; Hewitt et al., 1991). These proteins are cell-coded and cannot be precipitated from similarly radiolabelled control cells. Two of these polypeptides are significantly induced by HSV-2 infection (Hewitt et al., 1991; Macnab et al., 1992). This paper further characterises one of these polypeptides, U90, and addresses which properties distinguish the behaviour of U90 in tumour cells, whether there is an equivalent in control cells and whether U90 can be induced without HSV replication. U90 can be immunoprecipitated from radiolabelled human cells which are capable of extended passage in culture, as well as from human tumour cells, rodent tumour cells and cells of different lineages, e.g. epithelial, fibroblastic and lymphoid. Purification of U90 and the subsequent production of monospecific antibodies revealed, by western blotting, a homologue of 90 kDa in control cells. Western blotting shows that HSV can increase the amount of the U90 homologue in these normal cells. The absence of an immunoprecipitate of the U90 homologue from control cells is a result of the very short half-life (i.e. high turnover) of the protein in these cells (32.9 minutes) as opposed to tumour cells (about 13 hours).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of antitumor activity of vitamins K 1, K 2 and K 3 on human tumor cells by two (MTT and SRB) cell viability assays

Vitamin K (VK) congeners (VK 1, VK 2 and VK 3) have been used as antihemorrhagic agents, while VK 3 has also been found to inhibit growth in various rodent and human tumor cells. We have compared the antitumor activities of vitamin K 1, K 2, and K 3 against a panel of human cancer cell lines. For each test agent, a dose-response profile was generated by using an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and an SRB (sulforhodamine B) assay. Both assays yielded similar results. The respective ID 50 values of VK 3 in five hepatoma cell lines, HA59T, HA22T, PLC, HepG2, and Hep3B, of increasing differentiation state, were 42, 36, 28, 27, and 20 μM. For nasopharyngeal carcinoma (CG1), leukemia (U937), oral epidermoid carcinoma (KB), and breast carcinoma (BC-M1) cells, the ID 50 values of VK 3 were 26, 15, 25, and 33 μM, respectively. For all the above cells, the ID 50 values of VK 1 ranged from 6 to 9 mM, and the ID 50 values of VK 2 ranged from 1 to 2 mM. Thus, the relative potencies of antitumor activity of VK 3 compared to VK 2 and to VK 1 are about 60- and 300-fold, respectively. These results support the preference for use of VK 3 over VK 1 and VK 2 in cancer therapy.

Somatostatin receptor subtype gene expression in human and rodent tumors

Somatostatin (SRIF) analogues display anti-tumor properties believed to be mediated by specific cell surface somatostatin receptors (SSTR). SSTR subtypes have unique pharmacological properties, including specific GTP-binding protein coupling, ion channel regulation, and cAMP inhibition; therefoe, identification of isotypes expressed in tumor cells facilitates current efforts to design potent anti-tumor SRIF analogues. Human and rodent solid, transplantable tumors and tumor cell lines were examined for gene expression of SSTR1, SSTR2 and SSTR3 by reverse transcription of tumor mRNA and subsequent amplification of cDNA by the polymerase chain reaction, using SSTR subtype-specific oligonucleotide primers. SSTR2 mRNA transcripts were observed in all of the tumor cell lines examined. SSTR1 gene expression was seen in several human and rat types, and SSTR3 gene expression observed in two rodent tumor types. SSTR mRNA-positive tumors are expected to possess membrane-bound receptors which could potentially interact with anti-tumor SRIF analogues.

The antitumor activity of the platinum complex D-17872 is associated with tumor cell differentiation.

The novel cisplatin analogue D-17872 was studied for its anticancer activity using in vivo and in vitro preclinical models. The compound at the sublethal dose of 215 mg/kg (ca. 50% of the approximate LD50) induced no nephrotoxic effect strong enough to increase the blood urea level in rats. It had good in vivo antitumor efficacy against murine P388 (max. ILS: D-17872 132%, cisplatin 55%) and L1210 leukemia (max. ILS: D-17872 43%, cisplatin 38%), L5222 leukemia of the rat (max. ILS: D-17872 163%, cisplatin 163%) and murine B16 melanoma. Activity against P388 leukemia substantially exceeded that of cisplatin. Moreover, the M5076 reticulum cell sarcoma implanted into the subrenal capsule and the DMBA-induced mammary tumor of the rat were inhibited by D-17872 to a greater extent than by cisplatin (min. T/C: D-17872 -3%, cisplatin 11%). Using clonogenic microassays, D-17872 was active in vitro against a variety of human and rodent tumor cell lines, albeit at higher concentrations than cisplatin (IC50 values: D-17872 2.6-12.7 mumol/l, cisplatin 0.13-0.42 mumol/l). Apart from its cytotoxic action it was able to induce in vitro differentiation of the human HL-60 and K562 and of the murine M1-T22 cell lines, while cisplatin induced differentiation only in the HL-60 cell line. Thus D-17872 exhibited a pharmacological and toxicological profile different from that of the parent compound. The results suggest that induction of differentiation contributes to the antineoplastic efficacy of this novel cisplatin derivative.

Evidence for further heterogeneity of the receptors for neuropeptide-Y and peptide-YY in tumor cell lines derived from neural crest

The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.

Evidence for further heterogeneity of the receptors for neuropeptide-Y and peptide-YY in tumor cell lines derived from neural crest.

The expression and structure of the receptors for neuropeptide-Y (NPY) and peptide-YY (PYY) were studied in 16 human and rodent tumor cell lines derived from the neural crest by ligand binding and cross-linking techniques using [125I]Bolton-Hunter-NPY, [125I]PYY, and various forms of monoiodinated NPY and PYY. Although NPY-binding sites were observed in most of the tumor cells, PYY-binding sites were found only on the human neuroblastoma cell lines SMS-MSN, SMS-KAN, SK-N-MC, and MC-IXC and the human Ewing's sarcoma cell line SK-ES. The differential labeling of the NPY/PYY receptors on these cell lines suggests that the NPY/PYY receptors are more heterogeneous than previously described as the Y1, Y2, and Y3 receptor subtypes. Cross-linking studies demonstrate that the Y1 and Y2 receptors for NPY/PYY are structurally different (mol wt, 70 and 50 kilodaltons, respectively) and that the 70- and 50-kilodalton receptor proteins are coexpressed in certain tumor cell lines. This could explain at least in part why cell lines show a relative specificity for Y1/Y2 classification, observed as the inhibition by both C-terminal fragments and Y1-specific analogs on the NPY/PYY binding to membrane receptors. Collectively, the present study suggests further heterogeneity of the NPY/PYY receptors and the existence of multiple receptor proteins in the tumor cell lines derived from the neural crest.

Cytotoxicity of dual function nitrofurans in rodent and human tumor cells

The efficacy and selective hypoxic cell cytotoxicity of four dual function nitrofurans and two nitroimidazole-azividines was determined in human (A549, HT-29) and rodent (KHT/iv) tumor cells. All bioreductive compounds were found to be less effective at killing human than mouse tumor cells (∼ 2–6-fold). This reduced cytotoxicity in the human tumor cells occurred irrespective of the state of oxygenation. In addition, the degree of selective toxicity toward hypoxic cells or the cytotoxicity factor (CF), defined as the ratio of the surviving fraction in air to that under hypoxic conditions, was (a) greater for the nitroimidazole-aziridines than for the nitrofurans and (b) less in the human than the rodent tumor cell lines investigated. For example, CF values in A549 or HT-29 cells typically were 2–4-fold lower than those determined in KHT/iv cells. This reduction in the CF in the human cells resulted from a greater loss in the hypoxic toxicity than in the aerobic toxicity when compared with the rodent cells.

Analysis of integrin mRNA in human and rodent tumor cells

Several human and rodent tumor cell lines were examined for the presence of integrin mRNAs by dot- and Northern- blot analysis. All tumor cells tested expressed mRNAs for α5, αIIb, β1 and β3. The mRNA of β2 integrin was not detectable and that of α v integrin was found only in certain cells. Northern blotting was carried out in three selected tumor cell lines: Clone A, HEL and B16a. An apparent difference in the mRNA species coding for the αIIbβ3 integrin, but not for α5 and β1, was found. Our result suggests that alternative splicing of integrin genes may be one of the important mechanisms in regulating αIIbβ3 expression and function in different tumor cells.

Studies on the distribution of abnormal cells in cytological smears: VII. Cervical brush versus plastic and wooden spatulas

A model system of exfoliated normal human cervicovaginal squamous cells, exfoliated rodent tumor cells, and acellular, viscous, mucuslike material was used to investigate cell deposition on smear preparations made with three different instruments: plastic spatulas, wooden spatulas, and brush-tipped collectors. The total number of exfoliated cells and the total number of tumor cells present within the randomly distributed holes were then recorded for 41 smear preparations. For smears done with the brush, a total of 47,146 exfoliated cells were recorded; with wooden spatulas, 4517 cells; and with plastic spatulas, 7648 cells. When the brush was used, 6905 tumor cells were recorded. When wooden or plastic spatulas were applied, 563 and 1132 tumor cells were found, respectively. Thus, the brush yielded 12.2 and 6.1 times more tumor cells than plastic and wooden spatulas, respectively.