Contextual effects of transforming growth factor beta on the tumorigenicity of human colon carcinoma cells.

Transforming growth factor betas (TGF-betas) are a growth factor family with negative autocrine growth functions for most epithelial cells including colon carcinoma cell lines. Both type I (RI) and type II (RII) transmembrane TGF-beta receptors have been shown to be indispensable for TGF-beta-mediated cell growth regulation. Previous studies using different model systems have shown that both overexpression of TGF-beta1 and transfection of antisense TGF-beta1 to reduce TGF-beta1 expression could lead to increased tumorigenicity. These results are seemingly contradictory and suggest that effects of TGF-beta modulation on malignant properties of cancer cells may be contextual. This study addresses this issue using human colon carcinoma cells (CBS and FET) to determine the effects of modulation of the various components of the TGF-beta system on in vitro and in vivo growth properties in two independent isogenic models of colon carcinoma. Cells were stably transfected with a tetracycline-repressible RII expression vector (CBS4-RII), a tetracycline-repressible expression vector containing a truncated RII cDNA lacking the serine/threonine kinase domain (CBS4-deltaRII and FET6-deltaRII), or with a vector containing the TGF-beta1 cDNA (CBS4-beta1S and FET-beta1S). Expression of the truncated RII reduced TGF-beta sensitivity, whereas overexpression of RII increased TGF-beta sensitivity. TGF-beta overexpression did not affect TGF-beta response. In vivo tumorigenicity assays revealed that CBS4-RII cells had lower tumorigenicity than control cells, whereas CBS4-deltaRII and CBS4-beta1S had higher tumorigenicity than controls. The CBS4 cells are poorly tumorigenic in athymic mice, and the wild-type FET6 cells are nontumorigenic. FET6-deltaRII cells formed rapidly growing tumors, and FET-beta1S cells also formed tumors. These data illustrate the paradoxical tumor-promoting and -suppressing effects of TGF-beta signaling activity in two isogenic model systems from human colon carcinomas, thus demonstrating that the effects of modulation of TGF-beta expression or TGF-beta signaling capability affects malignancy in a contextual manner.

Control of type II transforming growth factor-beta receptor expression by integrin ligation.

Ectopic expression of the alpha5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of alpha5 integrin is due to induction of autocrine negative transforming growth factor-beta (TGF-beta) activity. MCF-7 breast cancer cells do not express either alpha5 integrin or type II TGF-beta receptor and hence are unable to generate TGF-beta signal transduction. Ectopic expression of alpha5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-beta receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in alpha5 transfectants by growth on fibronectin-coated plates. Induction of type II TGF-beta receptor expression also resulted in the generation of autocrine negative TGF-beta activity because colony formation was increased after TGF-beta neutralizing antibody treatment. Transient transfection with a TGF-beta promoter response element in tandem with a luciferase cDNA into cells stably transfected with alpha5 integrin resulted in basal promoter activities 5-10-fold higher than those of control cells. Moreover, when alpha5 transfectants were treated with a neutralizing antibody to either TGF-beta or integrin alpha5, this increased basal promoter activity was blocked. Autocrine TGF-beta activity also induced 3-fold higher endogenous fibronectin expression in alpha5 transfectants relative to that of control cells. Re-expression of type II receptor by alpha5 transfection also restored the ability of the cells to respond to exogenous TGF-beta and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-beta type II receptor expression can be controlled by alpha5beta1 ligation and integrin signal transduction. Moreover, TGF-beta and integrin signal transduction appear to cooperate in their tumor-suppressive functions.

Detection of poly(ADP-ribose) polymerase cleavage in response to treatment with topoisomerase I inhibitors: a potential surrogate end point to assess treatment effectiveness.

Cleavage of poly(ADP-ribose) polymerase (PARP) by caspases is a prominent characteristic of apoptosis or programmed cell death shown to be induced by topoisomerase (Topo) inhibitors. Because Topo I inhibitors have been shown to be effective in the treatment of some patients with colon cancer, we considered the possibility of using PARP cleavage as an early predictor of responsiveness to this class of agents. We show cleavage of PARP in response to treatment with Topo I inhibitors in colon cancer both in vitro and in vivo: (a) in vitro in SW480, HCT116, VACO5, VACO6, VACO8, VACO411, VACO425, and VACO451 human colon cancer cell lines treated with topotecan (TPT) or CPT-11; (b) in vivo in SW480, VACO451, and VRC5 colon cancer xenografts grown in athymic mice treated with TPT or CPT-11; and (c) in vivo in colon cancer samples from patients undergoing a Phase II clinical trial with CPT-11. Our results show a strong correlation between percentage of PARP cleavage and percentage of acridine orange-positive cells in colon cancer cell lines treated with 0.1 microM TPT for 24 and 48 h, confirming that PARP cleavage is a useful marker for programmed cell death in colon cancer cell lines. Results from experiments performed on colon cancer xenografts also show an association between PARP cleavage and response to treatment with TPT or CPT-11. The increase of PARP cleavage in xenografts and in clinical samples corresponding to treatment with Topo I inhibitors suggests that this procedure may have early predictive value to assess effectiveness of treatment. These results provide the basis for determining the validity of using PARP cleavage as an early marker of chemotherapeutic effectiveness in human samples.

Autocrine transforming growth factor alpha provides a growth advantage to malignant cells by facilitating re-entry into the cell cycle from suboptimal growth states.

CBS human colon carcinoma cells are poorly tumorigenic in athymic nude mice, whereas FET colon carcinoma cells are non-tumorigenic. Both cell lines have well differentiated properties in tissue culture. Transforming growth factor alpha (TGF-alpha) was ectopically expressed by stable transfection of a TGF-alpha cDNA under repressible tetracycline control. The TGF-alpha-transfected cells showed enhanced clonal initiation and shortened lag phase growth in tissue culture without an alteration in doubling time in exponential phase relative to untransfected cells. Furthermore, the TGF-alpha transfectants showed increased independence from exogenous growth factors in clonal growth assays and induction of DNA synthesis after release from quiescence. Growth factor independence was associated with sustained epidermal growth factor receptor activation in quiescent TGF-alpha-transfected cells and the requirement of exogenous insulin for stimulation of quiescent cells to re-enter the cell cycle. Higher cloning, reduced lag time in tissue, and the acquisition of growth factor independence for DNA synthesis without a change in doubling time of TGF-alpha-transfected cells indicate that autocrine TGF-alpha functions by facilitating re-entry into the cell cycle from sub-optimal growth states rather than promoting or controlling the proliferation of actively cycling cells. The modulation of growth regulation by autocrine TGF-alpha was associated with increased malignant properties as TGF-alpha transfectants showed increased tumorigenicity in athymic nude mice. The administration of tetracycline reversed the effects of TGF-alpha expression in these cells both in vivo and in vitro, indicating that the alterations of the biological properties were due to the expression of TGF-alpha. Since these cells are continuously grown in a completely chemically defined medium without serum supplementation, it was possible to assign the mechanism underlying the generation of growth factor independence to the replacement of a requirement for exogenous insulin in parental cells by autocrine TGF-alpha.

Expression of transforming growth factor-beta receptor type II and tumorigenicity in human breast adenocarcinoma MCF-7 cells.

To analyze transforming growth factor-beta (TGF-beta) response during MCF-7 cell progression, early passage (MCF-7E, < 200 passage) and late passage (MCF-7L, > 500 passage) cells were compared. MCF-7E cells showed an IC50 of approximately 10 ng/ml of TGF-beta1, whereas MCF-7L cells were insensitive. MCF-7E cells contained approximately threefold higher levels of TGF-beta receptor type II (TbetaRII) mRNA than MCF-7L, but their TbetaRI levels were similar. MCF-7E parental cells showed higher TbetaRII promoter activity than MCF-7L cells, which could be attributed to changes in Sp1 nuclear protein levels. Receptor cross-linking studies indicated that the cell surface receptor levels parallel mRNA levels in both cell lines. Limiting dilution clones of MCF-7E cells were established to determine the heterogeneity of TbetaRII expression in this cell line, and they showed varying degrees of TbetaRII expression. Fibronectin was induced at higher levels in cells expressing higher TbetaRII levels. All three TGF-beta isoforms were detected in limiting dilution clones and parental cells, but TGF-beta1 was more abundant relative to TGF-beta2 or 3, and no correlation between TGF-beta isoform profile with TGF-beta sensitivity was found. MCF-7L cells were tumorigenic and formed xenografts rapidly and progressively, whereas MCF-7E parental and limiting dilution clonal cells showed transient tumor formation followed by regression. These results indicate that decreased TbetaRII transcription in breast cancer cells leads to a loss of TbetaRII expression, resulting in cellular resistance to TGF-beta which contributes to escape from negative growth regulation and tumor progression.

A kinase-defective transforming growth factor-beta receptor type II is a dominant-negative regulator for human breast carcinoma MCF-7 cells.

The role of transforming growth factor (TGF)-beta type II receptor (T beta RII) in TGF-beta resistance and tumor progression is now well recognized. To test the effects of T beta RII loss in determining malignancy, we transfected a T beta RII-expressing, TGF-beta-sensitive, MCF-7 cell strain (ME24) with a tetracycline-repressible truncated T beta RII (kdT beta RII) construct lacking the cytoplasmic domain of the receptor. Transfection of kdT beta RII into parental ME24 cells (designated ME24t6 after transfection) resulted in high expression levels of kdT beta RII mRNA and cell surface protein which were reversible by tetracycline treatment. ME24t6 cells did not respond to exogenous TGF-beta 1 as measured by inhibition of proliferation or fibronectin (FN) induction, indicating that the truncated T beta RII acted as a dominant-negative inhibitor of both the growth inhibitory and extracellular matrix (ECM) stimulatory TGF-beta effects. Furthermore, inhibition of kdT beta RII expression by tetracycline treatment led to TGF-beta 1-mediated cell growth arrest in the G1 phase of cell cycle and to the accumulation of the hypophosphorylated form of retinoblastoma (Rb) protein. However, compared to parental ME24 cells, transfectants failed to show increased tumorigenicity, indicating that loss of T beta RII itself is not sufficient to account for differences in the malignant properties of T beta RII-expressing and non-expressing MCF-7 cell strains.

O6-methylguanine-DNA methyltransferase (MGMT) transfectants of a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-sensitive colon cancer cell line selectively repopulate heterogenous MGMT+/MGMT- xenografts after BCNU and O6-benzylguanine plus BCNU.

To evaluate the role of O6-alkylguanine-DNA alkyltransferase (AGT) in colon tumor chloroethylnitrosourea (CENU) resistance, AGT-deficient VACO 8 cells were transfected with a vector containing or lacking the human O6-methylguanine-DNA methyltransferase (MGMT) cDNA. VACO 8MGMT (V8MGMT) sublines possessed high levels of AGT activity in cell culture and were > 10-fold resistant to the CENU 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). V8MGMT cells, VACO 8neo cells, and mixtures of both were grown as xenografts in nude mice. MGMT expression in VACO 8 xenografts reflected the percentage of V8MGMT cells present in the tumor inoculum. Xenografts originally containing 0-10% V8MGMT cells were sensitive to BCNU, although partial resistance was observed as the percentage of V8MGMT cells increased. Tumors containing 30-100% V8MGMT cells were completely resistant to BCNU with no regressions and no growth delays. Pretreatment with O6-benzylguanine (BG) depleted tumor AGT activity for at least 6 h and sensitized xenografts containing 1 and 100% V8MGMT cells to BCNU. After BCNU or BG + BCNU, xenografts growing from inoculums containing as low as 0.1% V8MGMT cells had high AGT activities similar to that found in V8MGMT xenografts, with the majority of the cells expressing MGMT. These results provide evidence that MGMT expression influences both intrinsic and acquired colon tumor CENU resistance, that selective expansion of AGT+ colon tumor cells commonly occurs after CENU exposure, and that BG is effective in sensitizing colon tumors to CENUs, even when only a small fraction of the cells in a heterogeneous tumor express MGMT.

Topotecan increases topoisomerase IIalpha levels and sensitivity to treatment with etoposide in schedule-dependent process.

To elucidate the effect of topoisomerase (Topo) I inhibitors in the modulation of Topo II levels and sensitivity to Topo II-directed drugs, athymic mice bearing SW480 human colon cancer xenografts were treated with simultaneous, subsequent, or distant doses of topotecan and etoposide. This in vivo study demonstrates that simultaneous administration of topotecan and etoposide results in an antagonistic response. In contrast, inhibition of Topo I by topotecan results in a compensatory increase in Topo II alpha levels associated with increasing sensitivity of tumors to subsequent treatment with the Topo II inhibitor etoposide. Furthermore, we show that Topo II alpha levels decline 5 days after the last dose of topotecan, resulting in restoration of the original response of the xenografts to etoposide. Thus, this study emphasizes the critical role of schedule dependency to optimize the effectiveness of combination chemotherapy with Topo I and Topo II inhibitors.

Role of alpha 5 beta 1 integrin in determining malignant properties of colon carcinoma cells.

We characterized the expression of alpha 5 beta 1 integrin in two distinct phenotypes of colon carcinoma cell lines. Highly invasive colon cell lines (designated Group I cell lines) expressed higher levels of integrin alpha 5 beta 1 mRNA and protein than did poorly invasive colon cell lines (designated Group III cell lines). The relatively high expression of integrin alpha 5 beta 1 in Group I cell lines resulted in strong enhancement of cell adhesion to fibronectin (FN) tissue culture plates, whereas Group III cell lines showed little or no enhancement of cell adhesion by coating. There was no significant difference between Group I and Group III cell lines with respect to cell adhesion to laminin and collagen IV. Cell adhesion to FN in Group I cells was mainly mediated by integrin alpha 5 beta 1 because a monoclonal anti-alpha 5 subunit antibody could block cell adhesion to FN, whereas anti-alpha 2 and anti-alpha 3 antibodies had no effect on cell adhesion to FN. The divergence of alpha 5 beta 1 expression in these two distinct colon carcinoma phenotypes suggested that high expression of alpha 5 beta 1 might contribute to malignant progression in this model system. To test this hypothesis, GEO cells, a Group III cell line that did not express alpha 5 integrin, were transfected with the alpha 5 subunit. Stable transfection of alpha 5 sense cDNA into a typical GEO-limiting dilution clone led to the expression of alpha 5 subunit mRNA and cell surface alpha 5 beta 1 protein. The alpha 5 sense transfectants showed enhanced attachment to FN-coated plates and were more tumorigenic when the cells were injected into athymic nude mice. These results indicate that inappropriately high alpha 5 beta 1 integrin expression contributes to malignant progression in colon carcinoma.

Reduced expression of transforming growth factor beta type I receptor contributes to the malignancy of human colon carcinoma cells.

Transforming growth factor beta (TGFbeta) type I (RI) and type II (RII) receptors are essential for TGFbeta signal transduction. A human colon carcinoma cell line, designated GEO, is marginally responsive to TGFbeta and expresses a low level of RI mRNA relative to colon carcinoma cells, which are highly responsive to TGFbeta. Hence, the role of RI as a limiting factor for TGFbeta sensitivity and the contribution of low RI levels to the malignant phenotype of GEO cells were examined. Stable transfection of a tetracycline-regulatable rat RI cDNA increased TGFbeta1 binding to RI and resulted in increased growth inhibition by exogenous TGFbeta1. In contrast, although stable transfection of an RII expression vector into the same GEO cells increased TGFbeta1 binding to RII, growth inhibition by exogenous TGFbeta1 was not altered. This indicated that the low level of RI is a limiting factor for the growth-inhibitory effects of TGFbeta in GEO cells. RI-transfected cells were growth-arrested at a lower saturation density than GEO control cells. They also showed reduced growth and clonogenicity in plating efficiency and soft agarose assays, whereas RII-transfected cells did not show any differences from the NEO control cells in these assays. Tetracycline repressed RI expression in transfected cells and reversed the reduction in plating efficiency of RI-transfected clones, confirming that growth effects were due to increased RI expression in transfected cells. TGFbeta1 neutralizing antibody stimulated the proliferation of RI-transfected cells but had little effect on GEO control cells, indicating that increased autocrine-negative TGFbeta activity also resulted from increased RI expression. Tumorigenicity in athymic nude mice was significantly delayed in RI-transfected cells. These results indicate that low RI expression can be a limiting factor for response to exogenous TGFbeta, as well as TGFbeta autocrine-negative activity, and that reduction of RI expression can contribute to malignant progression.

Demonstration that mutation of the type II transforming growth factor beta receptor inactivates its tumor suppressor activity in replication error-positive colon carcinoma cells.

Escape from negative growth regulation by transforming growth factor beta (TGF-beta) as a result of the loss of TGF-beta type II receptor (RII) expression has been found to be associated with the replication error (RER) colorectal cancer genotype, which is characteristic of hereditary nonpolyposis colorectal cancers. The RER-positive HCT 116 colon carcinoma cell line was examined for RII mutations. A 1-base deletion was found within a sequence of 10 repeating adenines (nucleotides 709-718), which resulted in a frameshift mutation. Although it is reasonable to predict that the loss of RII function would be an important determinant of malignancy, the large number of potential mutations in cells of this phenotype raises the possibility that an RII mutation may not be a key event in the tumorigenic phenotype of these cells. One way to test directly the importance of RII mutations in determining the malignant phenotype would be to restore its expression. If restoration of expression leads to diminished tumorigenicity, it would indicate that RII mutation is an important determinant of malignancy in the RER phenotype. To determine whether restoration of RII would lead to reversal of malignancy in RER colon cancers, an RII expression vector was transfected into the HCT 116 cell line. RII stable clones showed mRNA and protein expression of transfected RII. The fibronectin mRNA level was increased by exogenous TGF-beta 1 treatment in a dose-dependent manner in RII-positive clones, whereas the control cells remained insensitive. The RII transfectants showed reduced clonogenicity in both monolayer culture and soft agarose. They were growth arrested at a lower saturation density than control cells. TGF-beta 1-neutralizing antibody stimulated the proliferation of RII-transfected but not control cells, indicating that the alterations in the growth parameters of the transfected cells were due to the acquisition of autocrine-negative activity. Tumorigenicity in athymic mice was reduced and delayed in RII transfectants. These results indicate that reconstitution of TGF-beta autocrine activity by reexpression of RII can reverse malignancy in RER colon cancers, thus verifying that the malignancy of hereditary nonpolyposis colorectal cancer can be directly associated with the loss of RII expression.

Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of epithelial cell growth. Human colon cancer cell lines with high rates of microsatellite instability were found to harbor mutations in the type II TGF-beta receptor (RII) gene. Eight such examples, due to three different mutations, were identified. The mutations were clustered within small repeated sequences in the RII gene, were accompanied by the absence of cell surface RII receptors, and were usually associated with small amounts of RII transcript. RII mutation, by inducing the escape of cells from TGF-beta-mediated growth control, links DNA repair defects with a specific pathway of tumor progression.

Expression of transforming growth factor beta type II receptor leads to reduced malignancy in human breast cancer MCF-7 cells.

The role of transforming growth factor (TGF) beta type II receptor in reversing the malignant phenotype of human breast cancer MCF-7 cells was examined. MCF-7 cells were insensitive to TGF beta 1 and expressed undetectable levels of cell surface TGF beta type I receptor (RI) and type II receptor (RII) by cross-linking with 125I-TGF beta 1. Stable transfection of a RII expression vector yielded 3 transfectants with varying levels of exogenous RII mRNA and protein levels. Expression of RII also increased TGF beta 1 binding to RI in all 3 clones. Proliferation of RII-positive clones was inhibited by exogenous TGF beta 1 in a dose-dependent manner, whereas the control clones remained TGF beta-insensitive. The RII transfectants were growth arrested in monolayer culture at saturation densities which were 41-66% of that of the Neo controls. They also showed reduced clonogenicity in soft-agarose. Tumorigenicity in ovariectomized, estrogen-supplemented nude mice was delayed in transfectants with low RII levels. Transfectants expressing high levels of RII showed a large reduction in tumorigenicity as well as a longer delay in tumor formation. Tumor growth was associated with loss of exogenous RII expression in transfectants. The results indicate that when systems for TGF beta signal transduction are intact, reconstitution of the TGF beta receptor system can lead to reversion of malignancy in cells lacking RII.

Sensitive enzymatic cycling assay for glutathione: measurements of glutathione content and its modulation by buthionine sulfoximine in vivo and in vitro in human colon cancer.

We have measured glutathione content in small tissue samples derived from biopsies of primary and metastatic human colon tumors and from colon cancer cell lines in tissue culture and xenografts in athymic mice. Measurements were performed using an enzymatic cycling assay designed to quantitate extremely low levels of glutathione (GSH) (down to 10(-14) mol) from perchlorate extracts of tissue samples weighing less than 1 mg wet weight. Glutathione was stable in these acid extracts for at least 6 months when stored at -80 degrees C. A survey of normal tissues in mice, rats, and some human tissues showed considerable variation in GSH content of different tissues but generally similar levels were identifiable for the same tissues from different species. The highest GSH level was 56.9 nmol/mg protein in rat liver and the lowest was 1.8 nmol/mg protein in rat skeletal muscle. High GSH levels were also determined in mouse and human liver, while low GSH levels were detected in mouse muscle. Human colon cancer cell lines showed slightly higher GSH levels than did colon cancer tumor samples obtained from biopsies. These studies revealed a marked inter-individual difference in tumor GSH content, as well as a difference in GSH content between tumor deposits at different metastatic sites in the same individual. These results indicate the importance of direct tumor measurements of GSH content in clinical trials designed to modulate tumor glutathione content to try to increase sensitivity to chemotherapy or radiation therapy. Buthionine sulfoximine, an inhibitor of gamma-glutamyl cysteine synthetase, was shown to produce almost complete depletion of GSH in four different human colon cancer cell lines in 24 h. Buthionine sulfoximine was also shown to be capable of producing drastic depletion of GSH in human colon cancer grown as xenografts in athymic animals.

Synergistic efficacy of O6-benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a human colon cancer xenograft completely resistant to BCNU alone.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (alkyltransferase) repairs cytotoxic DNA damage formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). High levels of this repair protein cause tumor drug resistance to nitrosoureas. To investigate the ability of a direct alkyltransferase inhibitor, O6-benzylguanine, to reverse the nitrosourea resistance of human colon cancer cells, we studied the VACO 6 cell line which has high alkyltransferase and is completely resistant to BCNU at maximal tolerated doses in the xenograft model. O6-Benzylguanine at 0.5 microgram/mL for 1 hr inactivated VACO 6 alkyltransferase by > 98% and reduced the IC50 of BCNU by 3- to 4-fold. Further analysis indicated that these two agents act in a highly synergistic fashion. In xenograft bearing athymic mice, dose-dependent depletion of hepatic and tumor alkyltransferase was noted. To maintain alkyltransferase depletion in the xenograft for at least 24 hr, two doses of 60 mg/kg O6-benzylguanine were given 1 hr prior and 7 hr after BCNU. Under these conditions, VACO 6 xenografts became responsive to BCNU with significant reductions (P < 0.001) in the tumor growth rate. The combination increased toxicity to the host, reducing the maximum tolerated dose of BCNU by approximately 50%. This study provides definitive evidence that high alkyltransferase activity is responsible for BCNU resistance in human colon cancer xenografts and that with careful drug scheduling, O6-benzylguanine can sensitize a tumor which is completely unresponsive to BCNU alone. Further studies which optimize the therapeutic index of BCNU and O6-benzylguanine in vivo will define the schedule to be used in broader preclinical studies.

Biological characterization of three new spontaneous mouse lymphomas and comparison with the leukemia P388.

Three spontaneous lymphomas of 23- to 24-month-old (AB/Jena X DBA/2 Jena)F1 females were serially transplanted into syngeneic hosts. They were histologically classified as malignant lymphoblastic lymphomas (ABDt2 and ABDt5) and as a malignant lymphoma of histiocytic type (ABDt6). All lymphomas disseminated into the liver, spleen, and pancreas, whereas ABDt2 and ABDt5 additionally infiltrated the bone marrow and leptomeninx. None of the tumors showed a leukemic growth form, but extreme granulocytosis was observed in leukemia P388-bearing mice. Since the lymphomas ABDt2 and ABDt5 expressed T-cell differentiation antigens Ly-1, Ly-2, and Ly-3, they are thought to be of T-cell origin. On the contrary to the lymphocytes of parental mouse strain ABDt2 and ABDt5 cells reacted with anti H-2.23 allosera. All tumors have been found to be sensitive to at least 5/7 clinically used anticancer drugs. But only ABDt2- and P388-bearing mice survived longer than 60 days after treatment with the potent anticancer drug 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone). Successful chemotherapy of both tumors was accompanied with resistance of the hosts against the second transplant of the same tumor. Summarizing the characteristics of the newly established transplantation tumors it is concluded that they can be recommended as screening models in the search for new antineoplastic agents.

Genetic monitoring of inbred mouse strains maintained at the Bhabha Atomic Research Centre, Bombay: serological typing for H-2 haplotypes and lymphocyte differentiation markers.

Seven inbred mouse strains, AKR, CBA, C3H, C57BL/6, C57BR/cd, DBA/2 and Swiss, maintained at the Bhabha Atomic Research Centre, Bombay (designated Bh) were monitored for compatibility with standard strains as well as for genetical homogeneity. For this purpose, five individual mice from each strain were typed serologically for H-2 class I and class II antigens and for lymphocyte differentiation markers, Thy-2, Lyt-1, Lyt-2, TL and Ly-10. In this latter testing, 15 inbred strains from the Institute of Immunology and Experimental Therapy, Wroclaw (designated Iiw) were included. Results revealed that none of the tested strains deviated from the reactivity pattern of standard strains described in the literature. No evidence of genetic contamination was found in any strain. In the course of these studies, several interesting observations were made: (1) Swiss/Bh strain is an intra H-2 recombinant, probably KdAbEbDbTlab, (2) C3H/Bh and C3H/Iiw are Ly-10a and Ly-10b, respectively (3) some (CBA, C3H, DBA/2 and GR/S) but not C58/Ly-2a strains cross reacted with high concentration of anti Lyt-2.2 monoclonal antibody HO.2.2.