Endometrial Adenocarcinoma Is the Most Frequently Diagnosed Female Gynecological Cancer

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Abstract:

Endometrial adenocarcinoma is the most frequently diagnosed female gynecological cancer predominantly seen in developed western countries. The higher incidence of spontaneously developing hormone dependent endometrial adenocarcinomas in 90% of virgin female BDII/Han rat strains made them established models to study and understand the development of endometrial cancers at genetic level. Though oxidative stress plays critical role in malignant transformation of endometrial adenocarcinoma, the previous CGH analysis revealed the amplification of Met gene centered around the chromosome 4q12-22 region and hence it could disrupt the signaling pathways essential for proliferation of normal cells. RUT 13 cell lines obtained from endometrial tissues of BDII/SPRD-Cu3 F2 progeny are used for preparing the metaphase chromosome slides and used as study samples. The aim of the present study is to detect the amplified Met gene on the chromosome 4 hybridized to fluorophore labeled Met-green probe by metaphase fluorescent in situ hybridization and the results show the amplified signals for Met gene on chromosome 4 in the fluorescent image obtained from the fluorescent microscopy.

Introduction:

The increasing incidence of endometrial cancer particularly takes place in industrialized countries and is thought to be due to the contributing factors such as aging of the population, hormonal imbalance, and obesity. Evaluation of the uterine cavity is triggered upon findings of endometrial adenocarcinomas present with postmenopausal bleeding related symptoms. (Jimenez et. al., 2008)

A unique model that is represented by the inbred rat strain BDII is used to investigate the development of endometrial adenocarcinoma (EAC) because of its genetic features which contributes to the development of the cancer. (Vollmer G, 2003). The tumor cell extracts for the present investigation were obtained by crossing the rats of BDII/Han strain with rats from non-susceptible inbred rat strain SPRD-Cu3. The F1 heterozygote (BDII/SPRD) males were backcrossed with female BDII rats which result in F2 progeny that developed endometrial adenocarcinomas.

Met gene on the other hand is a hepatocyte growth factor receptor (HGFR), an expressing gene also termed as Met proto-oncogene. Met oncogene possesses an anti-tumor activity in epithelial cells of the endometrium. The expression patterns of Met gene involving amplification are observed in endometrial adenocarcinomas (Walentinsson et. al., 2001).

The cytogenetic analysis of amplification of the Met gene is carried out by fluorescent in situ hybridization (FISH). FISH uses a fluorescently labelled probe which hybridizes specifically to the gene of interest and when observed under a fluorescent microscope shows coloured spots on the chromosomes where the gene is present. This technique is efficient in localizing required specific DNA sequences. Detection of the amplified genes provides valuable insights to understand the molecular pathogenesis of cancer and may act as molecular markers for diagnosis or prognosis of cancer if detected earlier. CGH data concludes that amplified genes involve in tumourogenicity including Met were located around chromosome 4q12-q22 (Walentinsson et al, 2001).

Aim:

The aim of the present study is to detect and analyse the amplification of Met gene in metaphase chromosomal spreads prepared from RUT 13 rat EAC cell cultures by Fluorescent in situ hybridization (FISH).

Materials and Methods:

Tumour Material:

Females of the inbred rat strain BDII/Han predisposed to endometrial adenocarcinoma were interbred with rats from the non-susceptible strains of SPRD-Cu3/Han. The F1, F2, and backcross progenies developed tumours, most of which were characteized as EAC RUT13 cell line. The material used for the present study is chromosomal metaphase spreads obtained after synchronizing metaphase RUT13 cells.

Cytogenetic Analysis by FISH:

The chromosome slides with metaphase spreads and the SpectrumGreen™direct labeled dUTP incorporated into DNA probe specific to the MET gene were prepared and provided by the supervisor. The FISH technique was performed according to the Nick Translation Kit (Cat No. 32-801300) protocol. The probe is precipitated as per the protocol except that the sonicated human placental DNA is replaced with nuclease free water. The probe and the metaphase chromosomal DNA on slide was denatured. The slide is dried and the denatured probe mix is added to the slide under dark conditions as labeled probe is light sensitive. Immediately the coverslip is applied and sealed with parafilm. The slide is placed in the humidified box (prepared by placing wet tissue underneath the surface holding slides), sealed the box with aluminium foil to protect them from light and placed in a hybridization incubator at 37oC for 19 hours for hybridization. After incubation the parafilm and the cover slip were removed and slide is immediately subjected to washing. The slide is air dried in darkness. Once the slide is dried DAPI II counterstain is added onto the hybridization area marked on the slide and coverslip is placed over it. The slides were viewed at 10X under fluorescence microscope using the filter for FITC (flourescien isothiocyanate) and then the lens is changed to 100X view to zoom into spread chromosome to check for green fluorescence.

Results and Discussion:

Figure 1: Fluorescent signals for Met gene (arrow) obtained from fluorescent microscope.

The FISH is performed on metaphase spread chromosomes from RUT13 cell lines to detect and localize the amplified Met gene by analyzing the result obtained from fluorescent microscope. The hybridization of Met-green probe to Met gene loci on homologous chromosomes show multiple fluorescent signals in the Figure 1.

The multiple signals show the amplified Met gene on a chromosome. As the Met gene is located on chromosome 4q12-22 (Walentinsson et al, 2001) , the result obtained shows that the amplification of Met gene is seen in RUT 13 cell lines on chromosome 4. The image only shows a single chromosome i.e. positive for Met gene amplification.

The CGH analysis (Levan et al, 2006) results had shown that a subset of hormone dependent type I endometrial adenocarcinomas of human origin demonstarted gain in one or both of the two regions that are homologous to human RNO 6q11~q16 and RNO4q13~21 chromosome segments 2p21~p25 and 7q21~q31, respectively. Met is one among the 15 genes selected for the CGH analysis located on the human region 7q21~q31. The fluorescent in situ hybridization analysis of recurrent gene amplification in human endometrial adenocarcinoma (Samuelson et al, 2008) had shown that the Met gene is amplified in both the BDII rat models as well as human endometrial adenocarcinomas suggesting it to be a candidate amplification target.

The BDII disease rat models which show reduced heterogeneity because of the limited gene pools compared to the more heterogeneous human tumours still serve as a better guiding model for understanding the molecular pathogenesis of a subset of human endometrial adenocarcinomas exhibiting homologous genetic aberrations.

Conclusion:

Thus we can conclude that RUT13, an endometrial adenocarcinoma cell lines accumulate amplified copies of MET gene on chromosome 4 which is analysed by FISH.

References:

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