Expression of the proto-oncogenes c-met and c-kit and their ligands, hepatocyte growth factor/scatter factor and stem cell factor, in SCLC cell lines and xenografts.

We examined a panel of 25 small cell lung cancer (SCLC) cell lines and nude mouse xenografts for expression of the proto-oncogenes c-met and c-kit, and for expression of the corresponding ligands, hepatocyte growth factor (HGF) (also known as scatter factor (SF)), and stem cell factor (SCF), respectively. Expression of mRNA was detected by Northern blotting, and c-met and c-kit protein expression was detected by Western blotting and immunocytochemistry. c-met and c-kit mRNA was expressed in 22 of the examined cell lines or xenografts, and coexpression of the two proto-oncogenes was observed in 20 tumours. Expression of c-met and c-kit protein paralleled in the mRNA expression. HGF/SF mRNA was expressed in two of the examined tumours, and only one of these also expressed the c-met proto-oncogene. SCF mRNA was expressed in 19 of the examined tumours, and in 18 of these coexpression of c-kit and SCF was present. The high percentage of SCLC tumours expressing c-met and c-kit indicates that these proto-oncogenes may have an important function in this disease. The rare coexpression of c-met and HGF/SF is evidence that an autocrine regulatory pathway is not present for this receptor/ligand system in SCLC, while the frequent coexpression of c-kit and SCF indicates that this receptor/ligand system may have an autocrine function in SCLC

Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines.

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II mRNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF-beta receptor proteins beta-glycan mRNA was rapidly down-regulated and this effect was sustained throughout the 24 h observation period. RI and RII mRNAs were slightly increased 24 h after treatment. In one cell line sensitive to growth inhibition by TGF-beta, 1 but lacking beta-glycan expression, and one cell line expressing only beta-glycan and thus TGF-beta 1 -resistant, no autoregulation of mRNA of either TGF-beta receptor was demonstrated. The results suggest that TGF-beta 1 regulates the expression of its receptors, in particular beta-glycan, and that this effect is dependent on co-expression of beta-glycan, RI and RII

Interaction between three subpopulations of Ehrlich carcinoma in mixed solid tumours in nude mice: evidence of contact domination.

Clonal interaction between three subpopulations of Ehrlich carcinoma were studied during growth as mixed solid tumours and as ascites tumours in immune-incompetent nude NMRI mice. The tumour cell lines differed in DNA content as determined by DNA flow cytometry (FCM). Tumour growth was evaluated by tumour growth curves including calculation of tumour volume doubling times, tumour weight on day 14, cell cycle times (per cent labelled mitoses) and cell cycle distributions (FCM). Two subpopulations (E1.15 and E1.95) showed nearly identical growth characteristics during both solid and ascites tumour growth. The third subpopulation (E1.80) grew more slowly. FCM on fine-needle tumour aspirates was used to determine the relative proportions of the cell populations in mixed solid tumours in which E1.95 showed a growth-dominating effect on E1.15. No such effect was demonstrated during single-cell tumour growth in ascitic fluid in which the cells had no intimate contact. Ascitic fluid from E1.95-bearing animals or radiation-killed E1.95 cells had no effect on the growth of E1.15, and no remote effect was seen when the two cell lines were growing in opposite flanks. This indicates that only viable E1.95 cells in close in vivo contact were able to induce growth inhibition of the E1.15 subpopulation. Both the E1.95 and the E1.15 cells dominated the E1.80 cells, but in these cases cell kinetic differences may have played a role as the E1.95 and the E1.15 lines grew faster than the E1.80. The E1.80 cell line had no dominating effect on the E1.15 or E1.95. It is concluded that non-immunologically mediated cellular dominance in heterogeneous tumours may contribute to the evolution of these tumours and may be involved in fundamental tumour biological phenomena

A short-term in vitro test for tumour sensitivity to adriamycin based on flow cytometric DNA analysis.

A new method to test the sensitivity of tumour cells to chemotherapy is presented. Tumour cells were incubated in vitro on agar, and drug-induced cell cycle perturbation was monitored by flow cytometric DNA analysis. In the present study the method was applied to monitor the effect of adriamycin on an adriamycin-sensitive Ehrlich ascites tumour and two adriamycin-resistant tumours. Adriamycin caused a dose-related accumulation of tumour cells in the G2 + M phase in the sensitive tumour. Drug concentrations greater than or equal to 100-fold higher were required to induce similar changes in the resistant tumours. The dose level causing maximum accumulation in the G2 + M phase is suggested as a parameter for quantifying the sensitivity. The results indicate that the method can be extended to sensitivity testing of human tumours

Effect of 17 beta-oestradiol on growth curves and flow cytometric DNA distribution of two human breast carcinomas grown in nude mice.

The effect of 17 beta-oestradiol on a "receptor positive" and on a "receptor negative" human breast carcinoma grown in nude mice was studied. Experimental growth data were used to determine the effect on tumour growth. Flow cytometric DNA analysis (FCM) performed on tumour tissue obtained by sequential fine-needle aspirations was used to estimate the effect on the cell cycle. In the receptor-positive breast carcinoma, oestradiol induced complete tumour regression and characteristic cell cycle changes. In the receptor-negative breast carcinoma, no changes in tumour growth and cell cycle distribution could be demonstrated following the treatment. The results indicate that the oestradiol-induced cell kill could be explained to some extent by the induction of polyploid cells, which eventually die. Since the cell cycle changes monitored by FCM in the receptor-positive breast carcinoma appeared prior to any reduction in the tumour size, the results suggest that FCM may prove a valuable method in the early detection of tumour response to hormone treatment in human breast cancer

Expression of cadherin and NCAM in human small cell lung cancer cell lines and xenografts.

Tumour cell adhesion, detachment and aggregation seem to play an important part in tumour invasion and metastasis, and numerous cell adhesion molecules are expressed by tumour cells. Several families of cell-cell adhesion molecules have been described, of which two groups are particularly well characterised, the cadherin family and the Ig superfamily member, neural cell adhesion molecule (NCAM). We investigated expression of these two adhesion molecule families in small cell lung cancer (SCLC) cell lines and xenografts by immunoblotting. Nineteen tumours established from 15 patients with SCLC were examined. All tumours but one expressed both cadherin and NCAM. The tumours expressed one, two or rarely three cadherin bands, and different combinations of two major isoforms of NCAM with M(r)'s of approximately 190,000 and 135,000. Polysialylation of NCAM, a feature characteristic of NCAM during embryonic development, which may play a role in connection with tumour invasion and metastasis, was found in 14/18 NCAM expressing SCLC tumours. Individual tumours grown as cell lines and as nude mouse xenografts showed no qualitative differences in cadherin or NCAM expression

Expression of transforming growth factor beta (TGF beta) receptors and expression of TGF beta 1, TGF beta 2 and TGF beta 3 in human small cell lung cancer cell lines.

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor beta receptors (TGF beta-r) and the expression of TGF beta mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg-1 protein with a KD ranging from 16 to 40 pM. TGF beta 1 binding to the receptors was confirmed by cross-linking TGF beta 1 to the TGF beta-r. Three classes of TGF beta-r were demonstrated, type I and type II receptors with M(r) = 65,000 and 90,000 and the betaglycan (type III) with M(r) = 280,000. Northern blotting showed expression of TGF beta 1 mRNA in ten, TGF beta 2 mRNA in two and TGF beta 3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGF beta-receptors and also produce TGF beta mRNAs

Growth suppression by transforming growth factor beta 1 of human small-cell lung cancer cell lines is associated with expression of the type II receptor.

Nine human small-cell lung cancer cell lines were treated with transforming growth factor beta 1 (TGF-beta 1). Seven of the cell lines expressed receptors for transforming growth factor beta (TGF-beta-r) in different combinations between the three human subtypes I, II and III, and two were receptor negative. Growth suppression was induced by TGF-beta 1 exclusively in the five cell lines expressing the type II receptor. For the first time growth suppression by TGF-beta 1 of a cell line expressing the type II receptor without coexpression of the type I receptor is reported. No effect on growth was observed in two cell lines expressing only type III receptor and in TGF-beta-r negative cell lines. In two cell lines expressing all three receptor types, growth suppression was accompanied by morphological changes. To evaluate the possible involvement of the retinoblastoma protein (pRb) in mediating the growth-suppressive effect of TGF-beta 1, the expression of functional pRb, as characterised by nuclear localisation, was examined by immunocytochemistry. Nuclear association of pRb was only seen in two of the five TGF-beta 1-responsive cell lines. These results indicate that in SCLC pRb is not required for mediation of TGF-beta 1-induced growth suppression

In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor.

Formation of metastasis is a multistep process involving attachment to the basement membrane, local proteolysis and migration into surrounding tissues, lymph or bloodstream. In the present study, we have analysed the correlation between in vitro invasion and presence of the epidermal growth factor receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16% of the cells added to the upper chamber were able to traverse the Matrigel membrane. Expression of several matrix metalloproteases (MMP), of tissue inhibitor of MMP (TIMP) and of cathepsin B was evaluated by immunoprecipitation, Western blot analysis and reverse transcriptase polymerase chain reaction (RT-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition of this antibody resulted in a significant reduction of the in vitro invasion in three selected EGFR-positive cell lines. Our results show that only EGFR-positive SCLC cell lines had the in vitro invasive phenotype, and it is therefore suggested that the EGFR might play an important role for the invasion potential of SCLC cell lines

Disaggregation of human solid tumours by combined mechanical and enzymatic methods.

Two combined mechanical and enzymatic disaggregation techniques and a simple mechanical disaggregation procedure were compared. The combined procedures involved a mechanical comminution of the tumour tissue followed by incubation in trypsin. In one method, the tissue was subjected to long-term trypsinization at 4 degrees C, and in the other procedure, repeated short-term trypsinization at 37 degrees C was applied. The results were compared in terms of the yield of viable cells, plating efficiency, the ability to produce tumours in nude mice, and DNA distribution as measured by flow cytometry. The combined techniques provided reproducible cell yields of 2-10 X 10(7) viable cells g-1 of tissue, whereas only a small number of tumour cells was produced by the mechanical method. DNA analysis demonstrated that only the long-term trypsinization procedure resulted in a representative cell yield from all the tumours tested