A microcell hybrid based elimination test to identify human chromosome 3 regions that antagonize tumor growth

Abstract

Deletions at multiple sites on the short arm of chr3 have been detected in many different types of human tumors. Each of the deleted regions is expected to harbor a tumor antagonizing/suppressor gene(s) (TSG). The earlier studies showed that the tumorigenicity of malignant cells could be suppressed by introducing a single human chr3. However monochromosomal hybrids system in tumorigenicity tests suffers the frequent loss of the introduced chromosome or its parts. We have therefore chosen the identification of the regions that are regularly lost in the course of mouse tumor passage.

In the first study we have shown that two monochromosomal A9 mouse fibrosarcoma microcell hybrids (MCHs) lost the introduced cytogenetically intact normal human chr3 during progressive growth in SCID mice. FISH showed only fragments translocated to mouse chromosomes. PCR analysis had revealed that markers spanning the 3p21-p24 region were regularly lost/absent in all of the 13 tumors derived from 5 MCHs. We suggested that our findings might be related to the postulated presence of TSG(s) in the 3p21-p24 as indicated by the frequent deletion of this region in solid tumors. We propose that the identification of tumor antagonizing genes may be supplemented by a microcell hybrids based approach, referred as the 'Elimination test' (Et).

In the following studies, by increasing the number of tumors (27 from 5 MCHs) and chr3 markers (53), we have identified a common eliminated region (CER) of about 7 cM at 3p21.3, between D3S1260 and D3S643/D3F15SF, and a frequently eliminated region (FER) at 3p14.2-p21, between D3S1235 and D3S1067, that was absent in 21 of the 27 tumors. Using a panel of 30 SCID tumors derived from 5 MCHs that carried chr3 from the same donor, we have defined within CER at it centromeric border, at 3p21.33 between D3S1029 and LRRC2 gene, a common eliminated region 1 (CER1) of about 2.4 Mb. A common eliminated region 2 (CER2) of about 1.1 Mb was mapped within CER at it telomeric border at 3p22 between RH94338 and SHGC-154057. Unlimited availability of tumor material and the utility of non-polymorphic DNA markers in high-resolution deletion mapping are important advantages of Et.

Subsequently, we have created human-human MCHs by transferring a normal human chr3 by microcell fusion into KH39 RCC cells that contained uniparentally disomic chr3. In SCID tumors derived from four of these human-human MCHs, we could identify the same eliminated regions on 3p (hCER1 and hFER) as in the human-murine model, but the centromeric border of hCER1 was shifted to the CCR2 gene away from the LRRC2 gene in human-murine CER1.

Later, from the one sub-clone of the previously studied and two new A9-based mono-chromosomal MCHs that carried cytogenetically normal human chr3 from three different donors, we generated 9 SCID mice tumors that remained positive for all of the 120 3p-specific PCR-markers tested ("chr3+" tumors). These tumors were analyzed by FISH chromosome painting and by RT-PCR for the expression of 14 human chr3p genes: 5 from CER1 (LIMD1, CCR1, CCR2, CCR3, CCR5), 5 from regions that are often homozygously deleted (HD) in carcinomas (ITGA4L, LUCA1, PTPRG, FHIT, DUTT1), and 4 other cancer-related genes (VHL, MLH1, TGM4, UBE1L).

Alone among the 14 genes examined, FHIT showed a tumor growth-associated change. It was expressed in MCH lines in vitro, but all of the 9 "chr3+" tumors analyzed lost the FHIT gene transcript. Further we have examined the human-murine "chr3+" and human-human SCID tumors by RT-PCR for the expression of 20 chr3p genes including 7 genes from CER1 at 3p21.33 and 9 candidate TSGs from the LUCA region at 3p21.31 defined by overlapping HDs in lung cancer cell lines. We have found that in addition to the FHIT gene, the LTF gene from CER1 has also lost its mRNA expression in "chr3+" tumors. Moreover, SCL38A3 from the LUCA region and DRR1 at 3p21-14.3, have down-regulated, reduced or lost, their expression after SCID passage.

In the human-human system the endogenous/exogenous FHIT transcript was maintained in the SCID tumors, but the level of the Fhit protein was reduced. In contrast to the loss of mRNA expression from human-murine MCHs during SCID passage, in the human-human system, three out of the 20 examined genes, LTF, LRRC2 (neighboring LTF), and SCL38A3 (from LUCA region), expressed in the donor mouse A9-based MCH line, were suppressed, with regard to mRNA expression, after the transfer of chr3 into recipient human tumor cell line KH39. DRR1 mRNA expression was reduced in all of the 6 analyzed tumors after SCID growth.

At least four of the 20 examined chr3p genes, FHIT, LTF, SCL38A3, and DRR1, showed in common a tumor-associated impairment: in human-murine system the loss or down-regulation of mRNA expression after growth in SCID mice and in human-human system the down-regulation of the expression (FHIT and DRR1) in tumors or loss of the expression (LTF and SCL38A3) already after the transfer of chr3 into the human tumor cells.